{"id":11016,"date":"2021-02-17T16:40:55","date_gmt":"2021-02-17T15:40:55","guid":{"rendered":"https:\/\/www.vtei.cz\/?p=11016"},"modified":"2024-07-17T11:16:52","modified_gmt":"2024-07-17T10:16:52","slug":"search-for-suitable-molecular-markers-for-species-differentiation-of-enterococci","status":"publish","type":"post","link":"https:\/\/www.vtei.cz\/en\/2021\/02\/search-for-suitable-molecular-markers-for-species-differentiation-of-enterococci\/","title":{"rendered":"Search for suitable molecular markers for species differentiation of enterococci"},"content":{"rendered":"

<\/i> This article is available in Czech only. For translation or more information on this topic, please contact author.<\/h4>\n

 <\/p>\n

Souhrn<\/h2>\n

Enterokoky pat\u0159\u00ed spolu se z\u00e1stupci druhu Escherichia coli <\/em>mezi tzv. indik\u00e1tory fek\u00e1ln\u00edho zne\u010di\u0161t\u011bn\u00ed, kter\u00e9 se pou\u017e\u00edvaj\u00ed p\u0159i hodnocen\u00ed mikrobiologick\u00e9 kvality koupac\u00edch vod. Jejich stanoven\u00ed se \u0159\u00edd\u00ed vyhl\u00e1\u0161kou MZ \u010cR \u010d. 238\/2011 Sb. a\u00a0prov\u00e1d\u00ed se kultiva\u010dn\u011b na selektivn\u00edch agarov\u00fdch m\u00e9di\u00edch. Je zn\u00e1mo, \u017ee ne v\u0161echny druhy enterokok\u016f jsou fek\u00e1ln\u00edho p\u016fvodu a\u00a0maj\u00ed tedy p\u0159\u00edmou souvislost s\u00a0fek\u00e1ln\u00edm zne\u010di\u0161t\u011bn\u00edm vody. Pro spr\u00e1vn\u00e9 hodnocen\u00ed kvality koupac\u00ed vody by bylo vhodn\u00e9 zn\u00e1t i\u00a0p\u016fvod t\u011bchto bakteri\u00ed. K\u00a0trasov\u00e1n\u00ed p\u016fvodu mikroorganism\u016f (tzv. MST, \u201emicrobial source tracking\u201c) se s\u00a0v\u00fdhodou pou\u017e\u00edv\u00e1 metod molekul\u00e1rn\u00ed biologie. Pomoc\u00ed PCR metody lze amplifikac\u00ed molekul\u00e1rn\u00edch marker\u016f (tj. specifick\u00fdch \u00fasek\u016f DNA) odli\u0161it r\u016fzn\u00e9 druhy enterokok\u016f. Publikac\u00ed zab\u00fdvaj\u00edc\u00ed se touto t\u00e9matikou je mnoho, nicm\u00e9n\u011b ve v\u011bt\u0161in\u011b studi\u00ed se k\u00a0druhov\u00e9mu rozli\u0161en\u00ed pou\u017e\u00edv\u00e1 pouze jeden marker, a\u00a0to p\u0159i pr\u00e1ci s\u00a0p\u0159\u00edrodn\u00edmi vzorky nemus\u00ed b\u00fdt v\u017edy dostate\u010dn\u00e9. Pro spolehlivou druhovou identifikaci u\u00a0p\u0159\u00edrodn\u00edch vzork\u016f by bylo v\u00fdhodn\u011bj\u0161\u00ed pou\u017e\u00edt kombinaci n\u011bkolika marker\u016f. Z\u00e1rove\u0148 by bylo vhodn\u00e9 aplikovat poznatky z\u00edskan\u00e9 z\u00a0experiment\u016f s\u00a0\u010dist\u00fdmi kulturami na p\u0159\u00edrodn\u00ed vzorky koupac\u00edch vod, a\u00a0to jak \u010dist\u00fdch, tak fek\u00e1ln\u011b zne\u010di\u0161t\u011bn\u00fdch. Pro praxi by byly d\u016fle\u017eit\u00e9 i\u00a0postupy umo\u017e\u0148uj\u00edc\u00ed PCR ze sm\u011bsn\u00e9ho p\u0159\u00edrodn\u00edho vzorku, tedy bez nutnosti p\u0159edkultivace na selektivn\u00edch m\u00e9di\u00edch, aby se maxim\u00e1ln\u011b zkr\u00e1tila doba, za kterou je zn\u00e1m v\u00fdsledek.<\/p>\n

C\u00edlem tohoto p\u0159\u00edsp\u011bvku je p\u0159ehledn\u011b shrnout publikovan\u00e9 molekul\u00e1rn\u00ed markery pro identifikaci jak enterokok\u016f, tak p\u0159\u00edbuzn\u00fdch mikroorganism\u016f, a\u00a0zhodnotit jejich mo\u017en\u00e9 pou\u017eit\u00ed v\u00a0mikrobiologii vody pro rychl\u00e9 za\u0159azen\u00ed z\u00e1stupc\u016f rodu Enterococcus<\/em> do druh\u016f p\u0159i anal\u00fdze p\u0159\u00edrodn\u00edch vzork\u016f.<\/p>\n

Charakteristick\u00e9 znaky enterokok\u016f<\/h2>\n

Enterokoky jsou gram pozitivn\u00ed, katal\u00e1za negativn\u00ed koky uspo\u0159\u00e1dan\u00e9 obvykle do kr\u00e1tk\u00fdch \u0159et\u00edzk\u016f. Jsou to chemoorganotrofov\u00e9, jejich\u017e hlavn\u00edm koncov\u00fdm produktem p\u0159i fermentaci cukr\u016f je kyselina ml\u00e9\u010dn\u00e1. A\u010dkoliv fenotypick\u00e9 a\u00a0biochemick\u00e9 odli\u0161en\u00ed enterokok\u016f od ostatn\u00edch bakteri\u00e1ln\u00edch druh\u016f je slo\u017eit\u00e9, mezi hlavn\u00ed znaky tohoto rodu pat\u0159\u00ed p\u0159\u00edtomnost D antigenu, tolerance k\u00a0vy\u0161\u0161\u00edm teplot\u00e1m (a\u017e 45 \u00b0C) i\u00a0schopnost r\u016fstu v\u00a0p\u0159\u00edtomnosti 6,5% NaCl. P\u0159i jejich identifikaci v\u00a0laborato\u0159i se pou\u017e\u00edvaj\u00ed selektivn\u00ed m\u00e9dia, na kter\u00fdch jsou eliminov\u00e1ny ostatn\u00ed bakteri\u00e1ln\u00ed druhy (nap\u0159. SB m\u00e9dium obsahuj\u00edc\u00ed azid sodn\u00fd pro potla\u010den\u00ed G- ty\u010dek).<\/p>\n

Taxonomicky pat\u0159\u00ed mezi Enterococcacae<\/em> a\u00a0v\u00a0sou\u010dasn\u00e9 dob\u011b zahrnuje tento rod 43 druh\u016f [1]. Typick\u00fdm z\u00e1stupcem je\u00a0E. faecalis.<\/em> Enterokoky se p\u0159irozen\u011b vyskytuj\u00ed v\u00a0tr\u00e1vic\u00edm traktu teplokrevn\u00fdch \u017eivo\u010dich\u016f v\u010detn\u011b \u010dlov\u011bka i\u00a0v\u00a0tr\u00e1vic\u00edm traktu n\u011bkter\u00fdch bezobratl\u00fdch (kor\u00fd\u0161i, \u0161neci) [2, 3] a\u00a0byly izolov\u00e1ny i\u00a0z\u00a0povrchov\u00fdch sladk\u00fdch i\u00a0slan\u00fdch vod, z\u00a0p\u016fdy \u010di z\u00a0rostlinn\u00e9 vegetace. N\u011bkter\u00e9 druhy (nap\u0159. E. faecalis, E. faecium<\/em>) jsou podm\u00edn\u011bn\u011b patogenn\u00ed.<\/p>\n

P\u0159\u00edtomnost enterokok\u016f ve vod\u011b m\u016f\u017ee m\u00edt r\u016fzn\u00fd p\u016fvod: bu\u010f jsou p\u0159irozenou sou\u010d\u00e1st\u00ed vodn\u00edho ekosyst\u00e9mu a\u00a0vyskytuj\u00ed se tam voln\u011b, nebo mohou b\u00fdt asociov\u00e1ny s\u00a0rostlinnou vegetac\u00ed (typicky E. mundtii<\/em>, E. casseliflavus<\/em>) [4, 5], nebo se zooplanktonem [6], p\u0159\u00edpadn\u011b se mohou do vody dostat z\u00a0p\u016fdy [7]. Dal\u0161\u00edm zdrojem mohou b\u00fdt odpadn\u00ed vody \u010di exkrementy \u017eivo\u010dich\u016f, a\u00a0to jak obratlovc\u016f, tak bezobratl\u00fdch. Za typick\u00e9 intestin\u00e1ln\u00ed druhy jsou pova\u017eov\u00e1ny E. faecalis, <\/em>E. faecium, E. durans, E. hirae <\/em>[8].<\/p>\n

Jak je z\u00a0v\u00fd\u0161e uveden\u00e9ho v\u00fd\u010dtu patrn\u00e9, ke spr\u00e1vn\u00e9 interpretaci zv\u00fd\u0161en\u00e9 hladiny enterokok\u016f ve vodn\u00edm prost\u0159ed\u00ed by bylo vhodn\u00e9 zn\u00e1t jejich p\u016fvod.<\/p>\n

Metody, kter\u00e9 se pou\u017e\u00edvaj\u00ed k\u00a0vystopov\u00e1n\u00ed p\u016fvodu ur\u010dit\u00fdch organism\u016f v\u00a0prost\u0159ed\u00ed, se souhrnn\u011b naz\u00fdvaj\u00ed \u201emicrobial source tracking\u201c (MST). Principem t\u011bchto metod je, \u017ee ur\u010dit\u00e9 druhy bakteri\u00ed jsou asociov\u00e1ny s\u00a0ur\u010dit\u00fdmi hostiteli \u010di prost\u0159ed\u00edm. U\u00a0enterokok\u016f jsou nap\u0159. druhy E. faecalis, E. faecium<\/em> pova\u017eov\u00e1ny za typick\u00e9 intestin\u00e1ln\u00ed druhy (a\u010dkoliv byly izolov\u00e1ny i\u00a0z\u00a0jin\u00fdch ekosyst\u00e9m\u016f) a\u00a0naopak E. mundtii,\u00a0<\/em>E. casseliflavus<\/em> jsou v\u011bt\u0161inou spojeny s\u00a0rostlinami [9, 10]. Jednou z\u00a0metod MST je PCR amplifikace ur\u010dit\u00fdch (definovan\u00fdch) \u00fasek\u016f DNA (tzv. molekul\u00e1rn\u00edch marker\u016f), na jejich\u017e z\u00e1klad\u011b je mo\u017en\u00e9 rozli\u0161it enterokoky do jednotliv\u00fdch druh\u016f. V\u00a0tomto sm\u011bru existuje zna\u010dn\u00e9 mno\u017estv\u00ed literatury t\u00fdkaj\u00edc\u00ed se nejen enterokok\u016f, ale i\u00a0jin\u00fdch rod\u016f bakteri\u00ed, a\u00a0je mo\u017en\u00e9 z\u00a0toho p\u0159i hled\u00e1n\u00ed vhodn\u00fdch molekul\u00e1rn\u00edch marker\u016f vych\u00e1zet.<\/p>\n

Molekul\u00e1rn\u00ed markery pro MST<\/h2>\n

Obecn\u00e1 charakteristika molekul\u00e1rn\u00edch marker\u016f<\/p>\n

Vhodn\u00fd molekul\u00e1rn\u00ed marker pro druhov\u00e9 rozli\u0161en\u00ed bakteri\u00ed by m\u011bl spl\u0148ovat n\u011bkolik krit\u00e9ri\u00ed:<\/p>\n

    \n
  1. m\u011bl by b\u00fdt \u0161iroce roz\u0161\u00ed\u0159en v genomech bakteri\u00ed, tj. ve v\u011bt\u0161in\u011b bakteri\u00ed by m\u011bl m\u00edt ortologn\u00ed gen,<\/li>\n
  2. na druhou stranu tak\u00e9 mus\u00ed obsahovat \u00faseky unik\u00e1tn\u00ed pro dan\u00fd druh bez mo\u017en\u00fdch paralogn\u00edch gen\u016f,<\/li>\n
  3. jejich velikosti mus\u00ed b\u00fdt dostate\u010dn\u00e9 pro mo\u017en\u00e1 porovn\u00e1v\u00e1n\u00ed, ale z\u00e1rove\u0148 ne p\u0159\u00edli\u0161 dlouh\u00e9, aby se daly cel\u00e9 sekvenovat,<\/li>\n
  4. sekvence mus\u00ed b\u00fdt dostate\u010dn\u011b reprezentativn\u00ed, aby postihla spolehliv\u011b charakteristiku genomu dan\u00e9ho druhu.<\/li>\n<\/ol>\n
    Tabulka 1. P\u0159ehled diskutovan\u00fdch molekul\u00e1rn\u00edch marker\u016f
    \nTable 1. Summary of discussed molecular markers<\/h5>\n\"\"<\/a>\n

    Markery diskutovan\u00e9 v\u00a0tomto p\u0159\u00edsp\u011bvku jsou shrnuty v\u00a0tabulce 1<\/em>. Zeigler a\u00a0kolektiv porovn\u00e1vali ve sv\u00e9 pr\u00e1ci 44 bakteri\u00e1ln\u00edch genom\u016f, reprezentuj\u00edc\u00edch 16 rod\u016f, za \u00fa\u010delem hled\u00e1n\u00ed takov\u00fdch genov\u00fdch oblast\u00ed, kter\u00e9 by mohly b\u00fdt pou\u017eity obecn\u011b na rozli\u0161en\u00ed bakteri\u00e1ln\u00edch druh\u016f. Z\u00a0celkov\u00fdch anal\u00fdz nakonec vyplynulo 8 r\u016fzn\u00fdch gen\u016f (genov\u00fdch oblast\u00ed), kter\u00e9 spl\u0148ovaly v\u00fd\u0161e uveden\u00e1 krit\u00e9ria [11]. Dan\u00e1 studie nezahrnovala genom enterokok\u016f, av\u0161ak pracovala mimo jin\u00e9 s\u00a0genomy 4 druh\u016f streptokok\u016f, mezi n\u011b\u017e se enterokoky p\u016fvodn\u011b \u0159adily. Z\u00a0navr\u017een\u00fdch 8\u00a0kandid\u00e1tn\u00edch gen\u016f byly 4 (recN<\/em>, glyA<\/em>, thdF<\/em>, uvrC<\/em>) zkoum\u00e1ny i\u00a0u\u00a0enterokok\u016f, a\u00a0to bu\u010f v\u00a0souvislosti s\u00a0tvorbou biofilmu (recN<\/em>), nebo resistence k\u00a0UV (uvrC<\/em>) [12, 13]. Geny glyA<\/em> a\u00a0thdF<\/em> byly pou\u017eity spolu s\u00a0jin\u00fdmi geny pro odli\u0161en\u00ed r\u016fzn\u00fdch bakteri\u00e1ln\u00edch patogen\u016f zp\u016fsobuj\u00edc\u00edch aliment\u00e1rn\u00ed infekce [14, 15]. Amplifikace recN<\/em> byla pou\u017eita i\u00a0pro odli\u0161en\u00ed n\u011bkolika druh\u016f streptokok\u016f [16]. Tento gen se tedy nab\u00edz\u00ed jako jeden z\u00a0n\u011bkolika vhodn\u00fdch kandid\u00e1t\u016f t\u00e9\u017e k\u00a0detekci enterokok\u016f.<\/p>\n

    Skupiny gen\u016f vhodn\u00fdch pro MST<\/h3>\n

    Geny k\u00f3duj\u00edc\u00ed komponenty transkrip\u010dn\u00edho a\u00a0transla\u010dn\u00edho apar\u00e1tu<\/h4>\n

    Geny, jejich\u017e produkty se pod\u00edl\u00ed na expresi genetick\u00e9 informace, tedy na transkripci a\u00a0translaci, spl\u0148uj\u00ed podm\u00ednku p\u0159\u00edtomnosti ortolog\u016f (tj. ve v\u0161ech mikroorganismech, kde jsou p\u0159\u00edtomn\u00e9, pln\u00ed stejnou funkci) a\u00a0z\u00e1rove\u0148 t\u00e9\u017e obsahuj\u00ed \u00faseky specifick\u00e9 pro dan\u00fd druh. Proto tak\u00e9 16S a\u00a023S rDNA byly mezi prvn\u00edmi kandid\u00e1ty na molekul\u00e1rn\u00ed markery pro MST. Pou\u017eit\u00edm r\u016fzn\u00fdch oblast\u00ed 16S rDNA jako biologick\u00e9ho markeru se zab\u00fdvalo mnoho skupin, a\u00a0to nejen u\u00a0enterokok\u016f\u00a0[17], ale v\u00a0souvislosti s\u00a0MST t\u00e9\u017e u\u00a0bakteri\u00ed rodu Bacteroides <\/em>[18, 19]. Bacteroides<\/em> tvo\u0159\u00ed v\u00fdznamnou \u010d\u00e1st st\u0159evn\u00ed mikrofl\u00f3ry teplokrevn\u00fdch \u017eivo\u010dich\u016f v\u010detn\u011b \u010dlov\u011bka, a\u00a0proto o\u00a0nich tak\u00e9 bylo uva\u017eov\u00e1no jako o\u00a0mo\u017en\u00fdch alternativn\u00edch indik\u00e1torech fek\u00e1ln\u00edho zne\u010di\u0161t\u011bn\u00ed [20, 21]. Na rozd\u00edl od E. coli<\/em> a\u00a0intestin\u00e1ln\u00edch enterokok\u016f jsou ale obt\u00ed\u017en\u011b kultivovateln\u00e9. Jejich z\u00e1stupci jsou druhov\u011b specifi\u010dt\u00ed pro ur\u010dit\u00e9 hostitele [22], \u010deho\u017e by bylo mo\u017en\u00e9 s\u00a0v\u00fdhodou vyu\u017e\u00edt pr\u00e1v\u011b pro MST. Sekvenace 16S rRNA pat\u0159\u00ed st\u00e1le mezi nejpou\u017e\u00edvan\u011bj\u0161\u00ed metody pro identifikaci druh\u016f enterokok\u016f, p\u0159esto\u017ee m\u00e1 svoje limity p\u0159i rozli\u0161en\u00ed bl\u00edzce p\u0159\u00edbuzn\u00fdch druh\u016f, zejm\u00e9na ze skupiny E. faecium<\/em> [23].<\/p>\n

    Tsiodras s\u00a0kolegy analyzoval sekvence genu 23S rDNA k\u00f3duj\u00edc\u00edho RNA velk\u00e9 podjednotky bakteri\u00e1ln\u00edho ribozomu. Pou\u017eili dom\u00e9nu V\u00a023S rDNA pro odli\u0161en\u00ed E. faecalis<\/em> od ostatn\u00edch druh\u016f enterokok\u016f [24].<\/p>\n

    Pozd\u011bji si badatel\u00e9 v\u0161imli, \u017ee zat\u00edmco geny pro 16S a\u00a023S rRNA jsou fylogeneticky velmi konzervovan\u00e9, intergenov\u00e9 oblasti vykazuj\u00ed v\u011bt\u0161\u00ed variabilitu a\u00a0byly by tedy pro odli\u0161en\u00ed druh\u016f vhodn\u011bj\u0161\u00ed. Na z\u00e1klad\u011b srovn\u00e1n\u00ed intergenov\u00e9 oblasti mezi 23S a\u00a016S rRNA Tyrell a\u00a0kolektiv mohli rozli\u0161it enterokoky do t\u0159\u00ed fylogenetick\u00fdch skupin (I, II, III, navr\u017een\u00fdch d\u0159\u00edve Facklamem a\u00a0Collinsem) [25, 26]. V\u00a0r\u00e1mci skupiny II bylo nav\u00edc i\u00a0mo\u017en\u00e9 odli\u0161it intestin\u00e1ln\u00ed druh E. faecium<\/em> od typicky p\u0159\u00edrodn\u00edho druhu E. casseliflavus<\/em> a\u00a0druhu E. mundtii<\/em>. V\u00fdhodou tohoto markeru je t\u00e9\u017e fakt, \u017ee pro amplifikaci se pro v\u0161echny druhy pou\u017e\u00edv\u00e1 jeden p\u00e1r primer\u016f a\u00a0jednotliv\u00e9 druhy se li\u0161\u00ed velikost\u00ed amplifikovan\u00e9ho \u00faseku (\u00fasek\u016f).<\/p>\n

    Obdobn\u011b Baele a\u00a0kolektiv pou\u017eili intergenovou oblast mezi geny pro tRNA k\u00a0odli\u0161en\u00ed dev\u00edti druh\u016f enterokok\u016f [27]. P\u0159i t\u011bchto experimentech pou\u017eili primery navr\u017een\u00e9 v\u00a0krajn\u00edch konzervovan\u00fdch oblastech tRNA gen\u016f, pro amplifikaci oblasti mezi t\u011bmito geny. To by mohlo umo\u017enit vyu\u017eit\u00ed t\u00e9to strategie u\u00a0mnoha bakteri\u00e1ln\u00edch druh\u016f, \u010demu\u017e nasv\u011bd\u010duje fakt, \u017ee obdobn\u00e1 genov\u00e1 oblast byla pou\u017eita pro druhov\u00e9 rozli\u0161en\u00ed u\u00a0streptokok\u016f [28], Acinetobacter<\/em> spp. [29], Listeria <\/em>spp. [30] a\u00a0stafylokok\u016f [31, 32].<\/p>\n

    Dal\u0161\u00edm slibn\u00fdm markerem je gen rpoA<\/em> k\u00f3duj\u00edc\u00ed \u03b1 podjednotku RNA polymer\u00e1zy. Bylo uk\u00e1z\u00e1no, \u017ee p\u0159i pou\u017eit\u00ed rpoA<\/em> spolu s\u00a0dal\u0161\u00edm markerem\u00a0\u2013 pheS<\/em> (gen pro fenylalanyl-tRNA synt\u00e1zu) je mo\u017en\u00e9 rozli\u0161it 30 druh\u016f enterokok\u016f [33]. Gen rpoA<\/em> byl t\u00e9\u017e mezi kandid\u00e1tn\u00edmi geny navr\u017een\u00fdmi Zeiglerem a\u00a0kol. nicm\u00e9n\u011b s\u00a0men\u0161\u00edm sk\u00f3re ne\u017e v\u00fd\u0161e zm\u00edn\u011bn\u00fdch vybran\u00fdch osm kandid\u00e1tn\u00edch gen\u016f. Zd\u00e1 se v\u0161ak, \u017ee pro enterokoky by mohl b\u00fdt na odli\u0161en\u00ed druh\u016f spolehliv\u011b pou\u017eit.<\/p>\n

    Podobn\u011b lze gen tuf <\/em>k\u00f3duj\u00edc\u00ed elonga\u010dn\u00ed faktor Tu vyu\u017e\u00edt pro odli\u0161en\u00ed enterokok\u016f od ostatn\u00edch klinicky v\u00fdznamn\u00fdch bakteri\u00ed [34]. Je t\u0159eba ale podotknout, \u017ee navr\u017een\u00e9 primery se v\u00e1zaly t\u00e9\u017e na sekvence ortologn\u00edch gen\u016f u\u00a0n\u011bkolika druh\u016f Listeria<\/em> spp. a\u00a0z\u00e1stupc\u016f rodu Abiotrophia<\/em>.<\/p>\n

    Geny k\u00f3duj\u00edc\u00ed dal\u0161\u00ed proteiny prim\u00e1rn\u00edho metabolismu<\/h4>\n

    Poroznost je v\u011bnov\u00e1na i\u00a0gen\u016fm k\u00f3duj\u00edc\u00edm proteiny prim\u00e1rn\u00edho metabolismu, p\u0159edev\u0161\u00edm gen\u016fm ddl<\/em>, sodA, atpA <\/em>a\u00a0cpn60<\/em>.<\/p>\n

    Gen ddl <\/em>k\u00f3duje D-ala:D-ala lig\u00e1zu nezbytnou p\u0159i synt\u00e9ze peptidoglykanu. Na rozd\u00edl od amplifikace intergenov\u00fdch oblast\u00ed, p\u0159i pou\u017eit\u00ed genu ddl<\/em>, byla pou\u017eita pro r\u016fzn\u00e9 druhy r\u016fzn\u00e1 dvojice primer\u016f. V\u00fdsledkem PCR byl potom bu\u010f specifick\u00fd namno\u017een\u00fd \u00fasek DNA odpov\u00eddaj\u00edc\u00ed genu dan\u00e9ho druhu, nebo \u017e\u00e1dn\u00fd produkt. Nev\u00fdhodou t\u00e9to metody je, \u017ee v\u00a0p\u0159\u00edpad\u011b v\u011bt\u0161\u00edho mno\u017estv\u00ed druh\u016f znamen\u00e1 identifikace t\u00edmto zp\u016fsobem velk\u00e9 mno\u017estv\u00ed jednotliv\u00fdch PCR reakc\u00ed. Pou\u017eit\u00edm ddl<\/em> jako markeru bylo mo\u017en\u00e9 rozli\u0161it \u010dty\u0159i druhy enterokok\u016f (E. faecium, <\/em>E. faecalis, E. gallinarum, E. casseliflavus<\/em>) [35]. Harwood a\u00a0kolektiv pozd\u011bji identifikovali ddl<\/em> \u00fasek, kter\u00fd byl specifick\u00fd pro odli\u0161en\u00ed E. faecalis<\/em> od ostatn\u00edch druh\u016f. Nicm\u00e9n\u011b odli\u0161en\u00ed druh\u00e9ho typick\u00e9ho intestin\u00e1ln\u00edho druhu E. faecium<\/em> bylo s\u00a0t\u00edmto markerem dost slo\u017eit\u00e9 a\u00a0\u010dasto ani nekorelovalo s\u00a0v\u00fdsledky biochemick\u00fdch test\u016f [36].<\/p>\n

    Na obdobn\u00e9m principu, tedy navr\u017een\u00ed dvojice primer\u016f specifick\u00fdch pro dan\u00fd druh v\u00a0r\u00e1mci jednoho genu navrhli Jackson a\u00a0kolektiv 23 p\u00e1r\u016f primer\u016f pro amplifikaci genu sodA<\/em> k\u00f3duj\u00edc\u00ed superoxiddismut\u00e1zu z\u00e1vislou na Mn [37]. Tyto primery byly pak pou\u017eity hromadn\u011b v\u00a0celkem 7 PCR reakc\u00edch (multiplex PCR). Layton a\u00a0kolektiv nav\u00edc optimalizovali pozici n\u011bkter\u00fdch primer\u016f, a\u00a0t\u00edm zv\u00fd\u0161ili jejich druhovou specifitu [38].<\/p>\n

    Produktem genu atpA<\/em> je alfa podjednotka ATP synt\u00e1zy, tedy enzymu zodpov\u011bdn\u00e9ho za synt\u00e9zu ATP. Bylo zji\u0161t\u011bno, \u017ee t\u00edmto markerem je mo\u017en\u00e9 rozli\u0161it v\u011bt\u0161inu druh\u016f enterokok\u016f. Pomoc\u00ed atpA<\/em> spolu s\u00a0rpoA <\/em>a\u00a0pheS<\/em> bylo mo\u017en\u00e9 je\u0161t\u011b zv\u00fd\u0161it spolehlivost druhov\u00e9ho ur\u010den\u00ed a\u00a0navrhnout kombinaci t\u00e9to trojice marker\u016f spolu s\u00a016S rDNA jako vhodn\u00e9 geny pro typizaci enterokok\u016f [33, 39].<\/p>\n

    Goh a\u00a0kolektiv vyu\u017eili degenerovan\u00fdch primer\u016f k\u00a0odli\u0161en\u00ed 17 druh\u016f enterokok\u016f na z\u00e1klad\u011b amplifikace genu pro chaperonin (cpn 60<\/em>) [40].<\/p>\n

    Geny pro faktory virulence a\u00a0antibiotick\u00e9 resistence<\/h4>\n

    Je na m\u00edst\u011b se domn\u00edvat, \u017ee intestin\u00e1ln\u00ed druhy enterokok\u016f se mohou od t\u011bch voln\u011b v\u00a0p\u0159\u00edrod\u011b \u017eij\u00edc\u00edch druh\u016f li\u0161it produkc\u00ed faktor\u016f virulence v\u010detn\u011b produkce adhesin\u016f. A\u010dkoliv se p\u016fvodn\u011b myslelo, \u017ee nap\u0159. povrchov\u00fd protein Esp s\u00a0funkc\u00ed adhesinu je specifick\u00fd pro izol\u00e1ty E. faecalis<\/em> poch\u00e1zej\u00edc\u00edch z\u00a0intestin\u00e1ln\u00edho traktu \u010dlov\u011bka [41], pozd\u011bji byl tento protein nalezen i\u00a0u\u00a0izol\u00e1t\u016f ze zv\u00ed\u0159at \u010di jin\u00fdch p\u0159\u00edrodn\u00edch vzork\u016f [42, 43].<\/p>\n

    P\u0159\u00edtomnost gen\u016f pro jin\u00e9 faktory virulence jako nap\u0159. pro povrchov\u00fd agreguj\u00edc\u00ed protein (asa 1<\/em>) \u010di gen pro \u017eelatin\u00e1zu (gel E)<\/em> se li\u0161ila i\u00a0u\u00a0klinick\u00fdch izol\u00e1t\u016f jednoho druhu [44]. Z\u00a0tohoto d\u016fvodu by mohlo b\u00fdt pou\u017eit\u00ed t\u011bchto gen\u016f jako marker\u016f nejednozna\u010dn\u00e9.<\/p>\n

    Typicky p\u0159\u00edrodn\u00ed druhy enterokok\u016f (E. casseliflavus<\/em> a\u00a0E. gallinarum<\/em>) jsou, na rozd\u00edl od druh\u016f izolovan\u00fdch z\u00a0klinick\u00fdch vzork\u016f, obecn\u011b citliv\u00e9 k\u00a0antibiotik\u016fm. Geny rezistence se \u010dasto pou\u017e\u00edvaj\u00ed jako markery pro odli\u0161en\u00ed bakteri\u00e1ln\u00edch druh\u016f i\u00a0rezistentn\u00edch versus citliv\u00fdch izol\u00e1t\u016f p\u0159edev\u0161\u00edm v\u00a0hum\u00e1nn\u00ed medic\u00edn\u011b. K\u00a0nejv\u011bt\u0161\u00edm sou\u010dasn\u00fdm probl\u00e9m\u016fm antibiotick\u00e9 rezistence u\u00a0enterokok\u016f pat\u0159\u00ed rezistence k\u00a0vankomycinu, kter\u00fd byl dlouho pod\u00e1v\u00e1n p\u0159i infekc\u00edch multirezistentn\u00edmi kmeny p\u0159edev\u0161\u00edm u\u00a0hospod\u00e1\u0159sk\u00fdch zv\u00ed\u0159at. Vankomycin inhibibuje synt\u00e9zu peptidoglykanu, na jeho\u017e synt\u00e9ze se mimo jin\u00fdch pod\u00edl\u00ed t\u00e9\u017e v\u00fd\u0161e diskutovan\u00e1 D-ala:D-ala lig\u00e1za (gen ddl<\/em>). Geny, jejich\u017e produkty jsou zodpov\u011bdn\u00e9 za rezistenci k\u00a0vankomycinu, vanA, vanB, vanC-1<\/em>, a\u00a0vanC-2<\/em> jsou p\u0159\u00edbuzn\u00e9 k\u00a0ddl <\/em>[35, 45, 46]. Proti vankomycin rezistentn\u00edm enterokok\u016fm se v\u00a0sou\u010dasn\u00e9 dob\u011b pou\u017e\u00edv\u00e1 antibiotikum linezolid inhibuj\u00edc\u00ed proteosynt\u00e9zu. Na resistenci k\u00a0linesolidu se pod\u00edl\u00ed produkty gen\u016f cfr<\/em> k\u00f3duj\u00edc\u00ed ribozom modifikuj\u00edc\u00ed enzymy a\u00a0optrA, <\/em>jeho\u017e produktem je ABC transporter [47]. Av\u0161ak pou\u017eit\u00ed gen\u016f rezistence pro rozli\u0161en\u00ed hum\u00e1nn\u00edch a\u00a0zv\u00ed\u0159ec\u00edch druh\u016f m\u00e1 svoje limity\u00a0\u2013 geny rezistence se mohou v\u00a0populaci rychle \u0161\u00ed\u0159it horizont\u00e1ln\u00edm p\u0159enosem nebo spolu s\u00a0transpozi\u010dn\u00edmi elementy a\u00a0jejich p\u0159enos \u00fazce souvis\u00ed se selek\u010dn\u00edm tlakem v\u00a0prost\u0159ed\u00ed. Nav\u00edc izol\u00e1ty enterokok\u016f hum\u00e1nn\u00edho p\u016fvodu mohou vykazovat zna\u010dn\u00e9 rozd\u00edly v\u00a0citlivosti k\u00a0antibiotik\u016fm i\u00a0v\u00a0r\u00e1mci\u00a0jednoho druhu a\u00a0jednoho prost\u0159ed\u00ed. Proto pou\u017eit\u00ed antibiotick\u00fdch gen\u016f jako marker\u016f m\u00e1 pravd\u011bpodobn\u011b jen omezen\u00e9 uplatn\u011bn\u00ed.<\/p>\n

    Z\u00e1v\u011br<\/h2>\n

    P\u0159\u00edsp\u011bvek shrnuje testovan\u00e9 a\u00a0publikovan\u00e9 molekul\u00e1rn\u00ed markery, kter\u00e9 byly nebo mohou b\u00fdt pou\u017eity pro druhov\u00e9 rozli\u0161en\u00ed enterokok\u016f, co\u017e by m\u011blo pomoci p\u0159i charakterizaci jejich p\u016fvodu v\u00a0koupac\u00edch vod\u00e1ch. V\u011bt\u0161ina studi\u00ed se zab\u00fdv\u00e1 v\u017edy jedn\u00edm vhodn\u00fdm genem, ale to m\u016f\u017ee b\u00fdt p\u0159i anal\u00fdze p\u0159\u00edrodn\u00edch nav\u00edc zne\u010di\u0161t\u011bn\u00fdch vzork\u016f nedosta\u010duj\u00edc\u00ed. Pro spolehliv\u00e9 za\u0159azen\u00ed enterokok\u016f do druh\u016f by bylo tedy lep\u0161\u00ed pou\u017e\u00edt kombinaci n\u011bkolika gen\u016f v\u010detn\u011b jednoho rodov\u011b specifick\u00e9ho obsahuj\u00edc\u00edho \u010d\u00e1st 16S rRNA. Naser navrhl pou\u017e\u00edt kombinaci gen\u016f rpoA<\/em>, pheS<\/em> atpA<\/em> a\u00a016S rDNA pro typizaci enterokok\u016f. Svoje v\u00fdsledky v\u0161ak ov\u011b\u0159oval na \u010dist\u00fdch kultur\u00e1ch. Bylo by tedy zaj\u00edmav\u00e9 p\u0159en\u00e9st tyto poznatky do praxe a\u00a0ov\u011b\u0159it je i\u00a0na p\u0159\u00edrodn\u00edch vzorc\u00edch koupac\u00edch vod. Krom\u011b toho by z\u00a0uveden\u00e9ho v\u00fd\u010dtu mohl b\u00fdt pro typizaci pou\u017eit je\u0161t\u011b n\u011bkter\u00fd z\u00a0gen\u016f navr\u017een\u00fdch Zeiglerem a\u00a0kolektivem jako nap\u0159. gen recN<\/em> p\u0159\u00edpadn\u011b glyA<\/em> nebo thdF. <\/em>Pro tyto geny by ale bylo nutn\u00e9 ud\u011blat je\u0161t\u011b podrobn\u011bj\u0161\u00ed in silico<\/em> sekvena\u010dn\u00ed anal\u00fdzy. Zd\u00e1 se, \u017ee krom\u011b k\u00f3duj\u00edc\u00edch oblast\u00ed, jsou zaj\u00edmav\u00e9 i\u00a0intergenov\u00e9 oblasti, jako nap\u0159. oblasti mezi geny pro tRNA. Jejich v\u00fdhodou je mo\u017enost pou\u017eit\u00ed univers\u00e1ln\u00edch primer\u016f v\u00a0konzervovan\u00fdch oblastech i\u00a0mo\u017enost srovn\u00e1n\u00ed s\u00a0jin\u00fdmi rody, u\u00a0nich\u017e byl ji\u017e tento p\u0159\u00edstup pou\u017eit.<\/p>\n

    V\u00fd\u010det zde prezentovan\u00fdch kandid\u00e1t\u016f nen\u00ed zdaleka \u00fapln\u00fd a\u00a0v\u00a0literatu\u0159e existuje je\u0161t\u011b \u0159ada dal\u0161\u00edch mo\u017en\u00fdch genov\u00fdch oblast\u00ed, kter\u00e9 by bylo mo\u017en\u00e9 pou\u017e\u00edt jako nap\u0159. dal\u0161\u00ed geny k\u00f3duj\u00edc\u00ed proteiny prim\u00e1rn\u00edho metabolismu, nebo naopak repetitivn\u00ed nic-nekoduj\u00edc\u00ed sekvence. N\u011bkter\u00e9 nebyly uvedeny z\u00a0praktick\u00e9ho\u00a0d\u016fvodu, nebo\u0165 se nehodily pro jednozna\u010dn\u00e9 odli\u0161en\u00ed p\u016fvodu zne\u010di\u0161t\u011bn\u00ed nebo k\u00a0nim nen\u00ed v\u00a0sou\u010dasn\u00e9 dob\u011b dostatek dat (nebylo na n\u011b odk\u00e1z\u00e1no ve v\u00edce studi\u00edch). Je zde v\u0161ak velk\u00fd potenci\u00e1l dal\u0161\u00edch mo\u017en\u00fdch kandid\u00e1t\u016f.<\/p>\n

    Pod\u011bkov\u00e1n\u00ed<\/h3>\n

    Tento p\u0159\u00edsp\u011bvek vznikl v\u00a0r\u00e1mci projektu TA\u00a0\u010cR TJ04000132 Vyu\u017eit\u00ed metod molekul\u00e1rn\u00ed biologie k\u00a0identifikaci zdroj\u016f zne\u010di\u0161t\u011bn\u00ed v\u00a0koupac\u00edch vod\u00e1ch. <\/em><\/p>\n","protected":false},"excerpt":{"rendered":"

    Enterococci, together with representatives of the species Escherichia coli, belong to the so-called indicators of faecal pollution, which are used in the evaluation of the microbiological quality of bathing waters. Their determination is governed by the Decree of the Ministry of Health No. 238\/2011 Coll. and performed by culture on selective agar media.<\/p>\n","protected":false},"author":8,"featured_media":11012,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[87,89],"tags":[2382,2385,2387,2386],"coauthors":[2359,231],"class_list":["post-11016","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-hydrochemistry-radioecology-microbiology","category-water-technology-water-supply-waste-water-treatment","tag-enterococcus","tag-faecal-contamination","tag-microbial-source-tracking-mst","tag-molecular-markers"],"acf":[],"_links":{"self":[{"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/posts\/11016","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/users\/8"}],"replies":[{"embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/comments?post=11016"}],"version-history":[{"count":4,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/posts\/11016\/revisions"}],"predecessor-version":[{"id":30613,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/posts\/11016\/revisions\/30613"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/media\/11012"}],"wp:attachment":[{"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/media?parent=11016"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/categories?post=11016"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/tags?post=11016"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/www.vtei.cz\/en\/wp-json\/wp\/v2\/coauthors?post=11016"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}