{"id":1729,"date":"2019-05-09T11:01:24","date_gmt":"2019-05-09T09:01:24","guid":{"rendered":"http:\/\/www.newslab.sk\/2019\/05\/09\/sekvenovanie-dna-v-laboratornej-diagnostike-bakterialnych-patogenov\/"},"modified":"2019-05-09T13:01:24","modified_gmt":"2019-05-09T11:01:24","slug":"dna-sequencing-in-laboratory-diagnostics-of-bacterial-pathogens","status":"publish","type":"post","link":"https:\/\/www.newslab.sk\/en\/dna-sequencing-in-laboratory-diagnostics-of-bacterial-pathogens\/","title":{"rendered":"DNA sequencing in laboratory diagnostics of bacterial pathogens"},"content":{"rendered":"

*All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf attachment at the end of the paper.<\/strong><\/span><\/p>\n

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V klinickom mikrobiologickom laborat\u00f3riu sa na identifik\u00e1ciu a typiz\u00e1ciu p\u00f4vodcov infek\u010dn\u00fdch ochoren\u00ed tradi\u010dne pou\u017e\u00edvaj\u00fa fenotypov\u00e9 diagnostick\u00e9 met\u00f3dy zalo\u017een\u00e9 na kultiv\u00e1cii mikroorganizmov, mikroskopick\u00fdch technik\u00e1ch a \u0161pecializovan\u00fdch postupoch, ako je biotypiz\u00e1cia, s\u00e9rotypiz\u00e1cia alebo stanovenie citlivosti na antibiotik\u00e1. Z\u00e1rove\u0148 v s\u00fa\u010dasnosti doch\u00e1dza k v\u00fdznamn\u00e9mu rozvoju technol\u00f3gi\u00ed sekvenovania DNA, \u010do umo\u017e\u0148uje v praktick\u00fdch mikrobiologick\u00fdch aplik\u00e1ci\u00e1ch \u010doraz viac vyu\u017e\u00edva\u0165 priame genotypov\u00e9 met\u00f3dy.<\/p>\n

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Sekvenovanie jednotliv\u00fdch g\u00e9nov <\/strong><\/p>\n

V taxon\u00f3mii bakt\u00e9ri\u00ed sa tradi\u010dne vyu\u017e\u00edva \u00fapln\u00e9 alebo \u010diasto\u010dn\u00e9 sekvenovanie g\u00e9nu pre 16S rRNA zodpovedn\u00e9ho za synt\u00e9zu RNA malej podjednotky riboz\u00f3mu. Ako prv\u00fd tento pr\u00edstup zaviedol Carl Woese, ktor\u00fd na z\u00e1klade porovnania sekvenci\u00ed rRNA v roku 1977 definoval Archea <\/em>ako samostatn\u00fa taxonomick\u00fa skupinu prokaryotov l\u00ed\u0161iacu sa od klasick\u00fdch bakt\u00e9ri\u00ed(1). 16S rRNA g\u00e9n m\u00e1 d\u013a\u017eku pribli\u017ene 1,5 kbp a nach\u00e1dzaj\u00fa sa na \u0148om konzervat\u00edvnej\u0161ie a variabilnej\u0161ie \u00faseky. To umo\u017e\u0148uje navrhn\u00fa\u0165 univerz\u00e1lne primery (pre v\u0161etky bakt\u00e9rie alebo skupinovo \u0161pecifick\u00e9), ktor\u00e9 sa pou\u017e\u00edvaj\u00fa na PCR a sekvenovanie. Pri anal\u00fdze je d\u00f4le\u017eit\u00e9, \u017ee z\u00edskan\u00e9 sekvencie mo\u017eno porovn\u00e1va\u0165 s datab\u00e1zami sekvenci\u00ed, a t\u00fdm vyhodnoti\u0165 pr\u00edbuznos\u0165 kme\u0148ov z\u00edskan\u00fdch v rozli\u010dn\u00fdch \u0161t\u00fadi\u00e1ch. Existuje nieko\u013eko relevantn\u00fdch datab\u00e1z riboz\u00f3mov\u00fdch sekvenci\u00ed: Ribosomal Database Project, Silva, GreenGenes. Datab\u00e1za Living Tree Project (http:\/\/www.arb-silva.de\/projects\/ living-tree\/) obsahuje sekvencie 16S and 23S rRNA g\u00e9nov typov\u00fdch kme\u0148ov validne op\u00edsan\u00fdch druhov bakt\u00e9ri\u00ed a arche\u00ed d\u00f4le\u017eit\u00fdch na taxonomick\u00e9 \u00fa\u010dely(2).<\/p>\n

Okrem nesporn\u00fdch v\u00fdhod m\u00e1 v\u0161ak identifik\u00e1cia bakt\u00e9ri\u00ed na z\u00e1klade 16S rRNA g\u00e9nu svoje limity. Prvou nev\u00fdhodou je vysok\u00e1 konzervat\u00edvnos\u0165 16S rRNA sekvenci\u00ed, preto v niektor\u00fdch pr\u00edpadoch nemo\u017eno jednozna\u010dne odl\u00ed\u0161i\u0165 kmene patriace do pr\u00edbuzn\u00fdch druhov, ako s\u00fa niektor\u00e9 streptokoky a enterobakt\u00e9rie, a tie\u017e nie je mo\u017en\u00e1 diskrimin\u00e1cia medzi r\u00f4znymi kme\u0148mi rovnak\u00e9ho druhu. Z\u00e1rove\u0148 gen\u00f3my bakt\u00e9ri\u00ed obsahuj\u00fa viacero k\u00f3pi\u00ed rRNA oper\u00f3nu (napr\u00edklad gen\u00f3m E. coli <\/em>m\u00e1 sedem rRNA oper\u00f3nov), ktor\u00e9 nie s\u00fa sekven\u010dne identick\u00e9, \u010do zni\u017euje presnos\u0165 pri stanoven\u00ed pr\u00edbuznosti kme\u0148ov(3). Okrem riboz\u00f3mov\u00fdch g\u00e9nov sa pri identifik\u00e1cii a typiz\u00e1cii bakt\u00e9ri\u00ed \u010dasto vyu\u017e\u00edva sekvenovanie g\u00e9nov k\u00f3duj\u00facich prote\u00edny. Tak\u00e9to sekvencie mo\u017eno porovn\u00e1va\u0165 na \u00farovni nukleotidov (vysok\u00e1 variabilita sp\u00f4soben\u00e1 degenerat\u00edvnos\u0165ou genetick\u00e9ho k\u00f3du) alebo na \u00farovni aminokysel\u00edn (vy\u0161\u0161ia konzervovanos\u0165 vhodn\u00e1 pri porovn\u00e1van\u00ed vzdialenej\u0161\u00edch tax\u00f3nov). Spa typiz\u00e1cia, zalo\u017een\u00e1 na sekvenovan\u00ed variabiln\u00fdch oblast\u00ed spa <\/em>g\u00e9nu k\u00f3duj\u00faceho stafylokokov\u00fd prote\u00edn A, je \u010dasto pou\u017e\u00edvan\u00e1 met\u00f3da na stanovenie pr\u00edbuznosti kme\u0148ov Staphylococcus aureus<\/em>(4).<\/p>\n

V roku 1998 bola na stanovenie pr\u00edbuznosti kme\u0148ov Neiseria meningitidis <\/em>zaveden\u00e1 met\u00f3da multilokusovej sekven\u010dnej typiz\u00e1cie (Multi-locus sequence typing, MLST<\/strong>) a nesk\u00f4r sa t\u00e1to met\u00f3da za\u010dala pou\u017e\u00edva\u0165 aj pri typiz\u00e1cii in\u00fdch klinicky v\u00fdznamn\u00fdch bakt\u00e9ri\u00ed(5). Met\u00f3da je zalo\u017een\u00e1 na sekvenovan\u00ed (naj\u010dastej\u0161ie) siedmich g\u00e9nov rozlo\u017een\u00fdch v r\u00f4znych \u010dastiach bakteri\u00e1lneho gen\u00f3mu. Cie\u013eov\u00e9 g\u00e9ny s\u00fa odli\u0161n\u00e9 pre r\u00f4zne bakteri\u00e1lne druhy, ale plat\u00ed z\u00e1sada, \u017ee sa vyberaj\u00fa g\u00e9ny z\u00e1kladn\u00e9ho metabolizmu (tzv. housekeeping g\u00e9ny), ktor\u00e9 predstavuj\u00fa najstabilnej\u0161iu \u010das\u0165 bakteri\u00e1lneho gen\u00f3mu.\u00a0 Pri bakt\u00e9ri\u00e1ch je \u010dast\u00fdm javom horizont\u00e1lny transfer g\u00e9nov medzi nepr\u00edbuzn\u00fdmi bakt\u00e9riami. Preto pri sekvenovan\u00ed iba jedn\u00e9ho lokusu, ktor\u00fd bol v predch\u00e1dzaj\u00facom obdob\u00ed predmetom g\u00e9nov\u00e9ho transferu, sa m\u00f4\u017eu dva kmene javi\u0165 ako identick\u00e9, ale sekvenovan\u00edm in\u00fdch \u010dast\u00ed ich gen\u00f3mov sa zist\u00ed, \u017ee tieto kmene nie s\u00fa pr\u00edbuzn\u00e9.<\/p>\n

Sekvenovan\u00edm v r\u00e1mci MLST sa preto ur\u010d\u00ed\/eliminuje vplyv horizont\u00e1lneho transferu g\u00e9nov medzi nepr\u00edbuzn\u00fdmi bakt\u00e9riami. Met\u00f3da MLST m\u00e1 oproti klasick\u00fdm typiza\u010dn\u00fdm technik\u00e1m (ako napr. PFGE) v\u00fdhodu v digit\u00e1lnom charaktere sekven\u010dn\u00fdch d\u00e1t. Pri vyhodnocovan\u00ed pr\u00edbuznosti kme\u0148ov sa postupuje tak, \u017ee sekvencie z jedn\u00e9ho lokusu (g\u00e9nu) sa navz\u00e1jom porovnaj\u00fa a jednotliv\u00e9 alely sa o\u010d\u00edsluj\u00fa v takom porad\u00ed, v akom sa z\u00edskali (t. j. sekvencia lokusu z prv\u00e9ho sekvenovan\u00e9ho kme\u0148a m\u00e1 \u010d\u00edslo 1, sekvencia nasleduj\u00faceho kme\u0148a l\u00ed\u0161iaca sa aspo\u0148 o jeden nukleotid m\u00e1 \u010d\u00edslo 2 at\u010f.). Na z\u00e1klade kombin\u00e1cie alel zo v\u0161etk\u00fdch lokusov (alelick\u00e9ho profilu) sa kme\u0148u prirad\u00ed v\u00fdsledn\u00e9 \u010d\u00edslo naz\u00fdvan\u00e9 sekven\u010dn\u00fd typ (ST). Vyhodnocovanie a uchov\u00e1vanie MLST d\u00e1t sa uskuto\u010d\u0148uje v datab\u00e1zach \u0161pecifick\u00fdch pre jednotliv\u00e9 druhy bakt\u00e9ri\u00ed. V\u00e4\u010d\u0161inu z t\u00fdchto datab\u00e1z mo\u017eno n\u00e1js\u0165 na str\u00e1nke http:\/\/pubmlst. org\/databases\/, na konci roku 2018 tam bolo deponovan\u00fdch 105 sch\u00e9m pre bakt\u00e9rie a 10 sch\u00e9m pre eukaryotick\u00e9 mikroorganizmy. Pri vyhodnocovan\u00ed podobnosti medzi izol\u00e1tmi sa vych\u00e1dza z alelick\u00fdch profilov. Pr\u00edbuzn\u00e9 kmene maj\u00fa rovnak\u00e9 ST alebo ST, ktor\u00e9 sa l\u00ed\u0161ia len v nieko\u013ek\u00fdch alel\u00e1ch, nepr\u00edbuzn\u00e9 kmene maj\u00fa sekvenciu odli\u0161n\u00fa vo v\u00e4\u010d\u0161ine lokusov. T\u00fdmto sp\u00f4sobom sa pr\u00edbuzn\u00e9 ST zhlukuj\u00fa do tzv. klon\u00e1lnych komplexov, ktor\u00e9 zahrnuj\u00fa s\u00fabor geneticky pr\u00edbuzn\u00fdch, ale nie \u00faplne identick\u00fdch bakt\u00e9ri\u00ed(5). Met\u00f3da MLST umo\u017e\u0148uje dobre rozl\u00ed\u0161i\u0165 kmene bakteri\u00e1lnych druhov s vysokou vn\u00fatrodruhovou variabilitou, napr. Neiseria meningitidis<\/em>, Campylobacter <\/em>sp., Streptococcus pneumoniae<\/em>, Cronobacter <\/em>sp. Na druhej strane existuj\u00fa patog\u00e9ny napr. Bacillus anthracis <\/em>alebo Yersinia pestis<\/em>, ktor\u00e9 sa vyzna\u010duj\u00fa vysokou genetickou homogenitou a pre ich typiz\u00e1ciu je met\u00f3da MLST m\u00e1lo diskriminat\u00edvna(5,6).<\/p>\n

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Celogen\u00f3mov\u00e9 sekvenovanie <\/strong><\/p>\n

Prv\u00fdm bakteri\u00e1lnym druhom so zn\u00e1mym gen\u00f3mom bol Haemophilus influenzae<\/em>, publikovan\u00fd v roku 1995(7). Na jeho sekvenovanie bol pou\u017eit\u00fd klasick\u00fd pr\u00edstup pr\u00edpravy gen\u00f3movej kni\u017enice v plazmidov\u00fdch vektoroch, sekvenovanie n\u00e1hodn\u00fdch fragmentov pomocou Sangerovej met\u00f3dy a bioinformatick\u00e9 skladanie z\u00edskan\u00fdch sekvenci\u00ed do v\u00fdslednej molekuly, preto bol tento postup pr\u00e1cny, pomal\u00fd a cenovo n\u00e1ro\u010dn\u00fd. V s\u00fa\u010dasnosti ho nahradili viacer\u00e9 met\u00f3dy sekvenovania novej gener\u00e1cie, ktor\u00e9 s\u00fa zalo\u017een\u00e9 na vysokokapacitnom paralelnom sekvenovan\u00ed DNA molek\u00fal. Tieto met\u00f3dy s\u00fa schopn\u00e9 generova\u0165 obrovsk\u00e9 mno\u017estv\u00e1 z\u00e1kladn\u00fdch sekven\u010dn\u00fdch d\u00e1t v kr\u00e1tkom \u010dase a pri n\u00edzkych n\u00e1kladoch na jednu sekvenovan\u00fa b\u00e1zu. Cena samotn\u00e9ho sekvenovania, ktor\u00e1 sa v s\u00fa\u010dasnosti pohybuje pod sumou 100 \u20ac za gen\u00f3m, u\u017e nie je limituj\u00facim faktorom, a tak sa celogen\u00f3mov\u00e9 sekvenovanie st\u00e1va dostupn\u00fdm aj pre mnoh\u00e9 rutinn\u00e9 laborat\u00f3ri\u00e1. Napr\u00edklad v USA s\u00fa od roku 2013 sekvenovan\u00e9 gen\u00f3my v\u0161etk\u00fdch izol\u00e1tov z hum\u00e1nnych klinick\u00fdch pr\u00edpadov L. monocytogenes<\/em>(8,9). D\u00f4le\u017eitou s\u00fa\u010das\u0165ou sekvenovania bakteri\u00e1lnych gen\u00f3mov je bioinformatick\u00e9 vyhodnocovanie z\u00edskan\u00fdch d\u00e1t. Vo v\u00e4\u010d\u0161ine s\u00fa\u010dasn\u00fdch next-gen techol\u00f3gi\u00ed je v\u00fdstupom ve\u013ek\u00e9 mno\u017estvo kr\u00e1tkych navz\u00e1jom sa prekr\u00fdvaj\u00facich sekven\u010dn\u00fdch \u010d\u00edtan\u00ed. Preto prv\u00fdm krokom pri anal\u00fdze d\u00e1t je skladanie (assembly) sekvenci\u00ed do \u010do najdlh\u0161\u00edch kontinu\u00e1lnych \u00fasekov (contigov). Zlo\u017een\u00e9 sekvencie, naj\u010dastej\u0161ie vo forme viacer\u00fdch nespojen\u00fdch line\u00e1rnych sekvenci\u00ed (whole genome contigs), sa v\u00a0klinickom mikrobiologickom laborat\u00f3riu m\u00f4\u017eu \u010falej vyu\u017ei\u0165 na presn\u00fa identifik\u00e1ciu p\u00f4vodcu ochorenia, stanovenie g\u00e9nov virulencie a rezistencie proti antibiotik\u00e1m a na ur\u010denie pr\u00edbuznosti kme\u0148ov pri epidemiologick\u00fdch \u0161t\u00fadi\u00e1ch. Pre spracovanie \u00fadajov z next-gen sekvenovania je potrebn\u00e9 ma\u0165 k dispoz\u00edcii bioinformatick\u00e9 n\u00e1stroje, ktor\u00e9 s\u00fa schopn\u00e9 spracova\u0165 ve\u013ek\u00e9 mno\u017estvo d\u00e1t vo ve\u013ek\u00fdch s\u00faboroch kme\u0148ov. Je \u017eiaduce, aby tieto n\u00e1stroje boli jednoduch\u00e9 na obsluhu a dostupn\u00e9 pre \u0161irok\u00fa odborn\u00fa verejnos\u0165 bez hlb\u0161\u00edch znalost\u00ed z informatiky a programovania. Jednou z mo\u017enost\u00ed pri vyhodnocovan\u00ed je vyu\u017eitie dostupn\u00fdch webov\u00fdch programov, napr\u00edklad pomocou aplik\u00e1ci\u00ed pr\u00edstupn\u00fdch na str\u00e1nke Center of genomic epidemiology (http:\/\/www. genomicepidemiology. org\/).<\/p>\n

Program SpeciesFinder vyh\u013ead\u00e1va v \u010diasto\u010dne sekvenovan\u00fdch gen\u00f3moch 16S rRNA g\u00e9ny a na z\u00e1klade najpr\u00edbuznej\u0161ej sekvencie zarad\u00ed kme\u0148 do bakteri\u00e1lneho druhu. In\u00fd sp\u00f4sob na druhov\u00fa identifik\u00e1ciu je K-mer- Finder, ktor\u00fd porovn\u00e1va v gen\u00f3mov\u00fdch sekvenci\u00e1ch pr\u00edtomnos\u0165 navz\u00e1jom sa prekr\u00fdvaj\u00facich k-merov s d\u013a\u017ekou 16 bp bez zoh\u013eadnenia ich lokaliz\u00e1cie na chromoz\u00f3me a kme\u0148 zarad\u00ed na z\u00e1klade najpr\u00edbuznej\u0161ieho kme\u0148a z datab\u00e1zy. Tento pr\u00edstup je ve\u013emi r\u00fdchly a dostato\u010dne presn\u00fd. \u010eal\u0161ie programy umo\u017e\u0148uj\u00fa zisti\u0165 z celogen\u00f3mov\u00fdch sekvenci\u00ed niektor\u00e9 d\u00f4le\u017eit\u00e9 vlastnosti kme\u0148ov: napr. MLST, spa typ, s\u00e9rotyp, pr\u00edtomnos\u0165 plazmidov, g\u00e9nov virulencie a g\u00e9nov rezistencie proti antibiotik\u00e1m(10). In\u00fdm sp\u00f4sobom porovn\u00e1vania celogen\u00f3mov\u00fdch sekvenci\u00ed je vyu\u017eitie pr\u00edstupu MLST, tzv. celogen\u00f3mov\u00e9 MLST (wg- MLST) pomocou softv\u00e9rov\u00e9ho n\u00e1stroja BIGSdb (Bacterial Isolate Genome Sequence Database)(6). Tento program extrahuje z celogen\u00f3mov\u00fdch sekvenci\u00ed bakteri\u00e1lnych izol\u00e1tov sekvencie jednotliv\u00fdch g\u00e9nov, sekvenci\u00e1m prirad\u00ed \u010d\u00edsla alel podobn\u00fdm sp\u00f4sobom ako v klasickej MLST, a potom porovn\u00e1va kmene na z\u00e1klade zhody alebo rozdielov v alel\u00e1ch jednotliv\u00fdch g\u00e9nov.<\/p>\n

Program je vytvoren\u00fd hierarchicky a mo\u017eno v \u0148om \u013eubovolne zvoli\u0165 s\u00fabor g\u00e9nov, ktor\u00e9 sa pou\u017eij\u00fa pri anal\u00fdze. Rovnak\u00fa datab\u00e1zu preto mo\u017eno vyu\u017ei\u0165 na druhov\u00fa identifik\u00e1ciu p\u00f4vodcu ochorenia pomocou anal\u00fdzy riboz\u00f3mov\u00fdch g\u00e9nov (rMLST), na zaradenie kme\u0148ov do sekven\u010dn\u00fdch typov a klon\u00e1lnych komplexov pomocou klasickej MLST aj na typiz\u00e1ciu kme\u0148ov s vysokou diskrimin\u00e1ciou (napr. pri sledovan\u00ed v\u00fdvoja epid\u00e9mi\u00ed v r\u00e1mci jednej nemocnice), ke\u010f sa porovn\u00e1vaj\u00fa sekvencie v\u0161etk\u00fdch spolo\u010dn\u00fdch g\u00e9nov (wgMLST)(6,11). Komplexn\u00fdm n\u00e1strojom na anal\u00fdzu d\u00e1t z celogen\u00f3mov\u00e9ho sekvenovania prokaryotick\u00fdch organizmov je webov\u00e1 aplik\u00e1cia PATRIC (The Pathosystems Resource Integration Center) (12). PATRIC umo\u017e\u0148uje online anal\u00fdzu bakteri\u00e1lnych gen\u00f3mov jednotliv\u00fdch pou\u017e\u00edvate\u013eov a ich porovn\u00e1vanie s verejne dostupn\u00fdmi \u00fadajmi. Program je prim\u00e1rne zameran\u00fd na patog\u00e9ny, preto m\u00e1 ve\u013ek\u00fa perspekt\u00edvu vyu\u017eitia v klinickom v\u00fdskume. Jednou z v\u00fdznamn\u00fdch vlastnost\u00ed je vyh\u013ead\u00e1va\u010d g\u00e9nov rezistencie proti antibiotik\u00e1m, ktor\u00fd vyu\u017e\u00edva datab\u00e1zy so zn\u00e1mymi mechanizmami rezistencie ako CARD (Comprehensive Antibiotic Resistance Database) a NDARO (National Database of Antibiotic Resistant Organisms). PATRIC \u010falej umo\u017e\u0148uje identifikova\u0165 g\u00e9ny k\u00f3duj\u00face virulen\u010dn\u00e9 faktory a transport\u00e9ry, modelova\u0165 metabolick\u00e9 dr\u00e1hy a porovn\u00e1va\u0165 prote\u00f3my jednotliv\u00fdch organizmov. Na identifik\u00e1ciu bakt\u00e9ri\u00ed mo\u017eno vyu\u017ei\u0165 najbli\u017e\u0161ieho pr\u00edbuzn\u00e9ho organizmu v datab\u00e1ze a vyhodnoti\u0165 fylogenetick\u00e9 vz\u0165ahy v r\u00e1mci vlastnej zbierky mikroorganizmov. Komplexnos\u0165 syst\u00e9mu PATRIC dop\u013a\u0148aj\u00fa aj aplik\u00e1cie umo\u017e\u0148uj\u00face z\u00e1kladn\u00e9 spracovanie sekven\u010dn\u00fdch d\u00e1t, t. j. skladanie (assembly) kr\u00e1tkych \u010d\u00edtan\u00ed do contigov a anot\u00e1cia sekvenci\u00ed.<\/p>\n

 <\/p>\n

Stanovenie citlivosti na antibiotik\u00e1 <\/strong><\/p>\n

Stanovenie citlivosti p\u00f4vodcu ochorenia k antimikrobi\u00e1lnym l\u00e1tkam je jednou zo z\u00e1kladn\u00fdch \u00faloh klinick\u00e9ho mikrobiologick\u00e9ho laborat\u00f3ria. V s\u00fa\u010dasnosti sa testovanie antibiotickej citlivosti ur\u010duje takmer v\u00fdhradne fenotypov\u00fdmi met\u00f3dami na z\u00e1klade rastu bakt\u00e9ri\u00ed v pr\u00edtomnosti antibakteri\u00e1lnych l\u00e1tok. Z\u00e1rove\u0148 v\u0161ak s\u00fa\u010dasn\u00e9 technol\u00f3gie umo\u017e\u0148uj\u00fa odvodi\u0165 rezistenciu kme\u0148ov pomocou vyh\u013ead\u00e1vania g\u00e9nov rezistencie v bakteri\u00e1lnej DNA. Identifik\u00e1cia g\u00e9nov rezistencie je vhodn\u00e1 aj na overenie citlivosti kme\u0148ov, ktor\u00e1 bola ur\u010den\u00e1 fenotypovo, a tie\u017e na identifik\u00e1ciu kme\u0148ov so slabou expresiou v podmienkach in vitro<\/em>. Detekcia t\u00fdchto g\u00e9nov sa tradi\u010dne rob\u00ed pomocou met\u00f3d PCR alebo DNA microarray(13).<\/p>\n

V\u00fdhodou anal\u00fdzy celogen\u00f3mov\u00fdch sekvenci\u00ed je, \u017ee v princ\u00edpe poskytuj\u00fa v\u0161etky \u00fadaje na ur\u010denie molekul\u00e1rnych mechanizmov antimikrobi\u00e1lnej rezistencie, a to aj bez predch\u00e1dzaj\u00facej znalosti fenotypu. Na predikciu g\u00e9nov rezistencie existuj\u00fa viacer\u00e9 \u0161pecializovan\u00e9 datab\u00e1zy. Jednou z najpou\u017e\u00edvanej\u0161\u00edch je datab\u00e1za ARG-ANOT (Antibiotic Resistance Gene-ANNOTation), ktor\u00fa mo\u017eno stiahnu\u0165 a vyu\u017e\u00edva\u0165 pomocou lok\u00e1lneho softv\u00e9ru na anal\u00fdzu sekvenci\u00ed( 14). Druhou mo\u017enos\u0165ou je vyu\u017eitie webov\u00fdch aplik\u00e1ci\u00ed, ako je ResFiner (15) alebo CARD(16), ktor\u00e9 s\u00fa pravideln\u00e9 aktualizovan\u00e9. Stanovenie rezistencie priamo z bakteri\u00e1lnych celogen\u00f3mov\u00fdch sekvenci\u00ed zatia\u013e st\u00e1le komplikuj\u00fa nedostato\u010dn\u00e9 vedomosti o v\u0161etk\u00fdch genetick\u00fdch zmen\u00e1ch ved\u00facich k zn\u00ed\u017eenej citlivosti bakteri\u00e1lnych kme\u0148ov na antibiotik\u00e1.<\/p>\n

Najv\u00e4\u010d\u0161\u00edm probl\u00e9mom pri rutinnej implement\u00e1cii WGS je aj nedostatok automatizovan\u00fdch, pou\u017e\u00edvate\u013esky pr\u00edvetiv\u00fdch interpreta\u010dn\u00fdch n\u00e1strojov, pomocou ktor\u00fdch by bolo mo\u017en\u00e9 r\u00fdchlo a jednoducho poskytn\u00fa\u0165 klinicky v\u00fdznamn\u00e9 inform\u00e1cie(17). R\u00fdchly v\u00fdvoj technol\u00f3gi\u00ed v tejto oblasti je predpokladom efekt\u00edvneho vyu\u017eitia v bl\u00edzkej bud\u00facnosti.<\/p>\n

 <\/p>\n

Z\u00e1ver <\/strong><\/p>\n

Techniky celogen\u00f3mov\u00e9ho sekvenovania maj\u00fa ve\u013ek\u00fd potenci\u00e1l sta\u0165 sa pevnou s\u00fa\u010das\u0165ou rutinn\u00fdch vy\u0161etren\u00ed v klinickej mikrobiol\u00f3gii, preto\u017ee umo\u017e\u0148uj\u00fa detailn\u00fa anal\u00fdzu p\u00f4vodcov infek\u010dn\u00fdch ochoren\u00ed pomocou univerz\u00e1lneho pr\u00edstupu. Ich v\u00fdhodou je aj mo\u017enos\u0165 identifik\u00e1cie patog\u00e9nov v zmesov\u00fdch vzork\u00e1ch bez predch\u00e1dzaj\u00facej kultiv\u00e1cie. Okrem identifik\u00e1cie a typiz\u00e1cie mikroorganizmov s\u00fa tieto techniky principi\u00e1lne vhodn\u00e9 na ur\u010denie rezistencie proti antibiotik\u00e1m, aj ke\u010f v s\u00fa\u010dasnosti e\u0161te nemo\u017eno jednozna\u010dne odvodi\u0165 fenotyp mikroorganizmu zo zn\u00e1mej gen\u00f3movej sekvencie. V bud\u00facnosti je predpokladom \u010fal\u0161ie zlep\u0161ovanie technol\u00f3gi\u00ed sekvenovania DNA, skr\u00e1tenie \u010dasu a zn\u00ed\u017eenie ceny anal\u00fdz, tak\u017ee tieto postupy bud\u00fa \u010doraz viac nahr\u00e1dza\u0165 tradi\u010dn\u00e9 met\u00f3dy z\u00e1visl\u00e9 od kultiv\u00e1cie, pou\u017e\u00edvan\u00e9 v klinick\u00fdch laborat\u00f3ri\u00e1ch.<\/p>\n

 <\/p>\n

Grantov\u00e1 podpora: <\/em><\/strong>T\u00e1to publik\u00e1cia vznikla s podporou grantov APVV-16-0168, APVV-16-0119 a projektu Biomakro 2 (ITMS 26240120027).<\/em><\/p>\n

 <\/p>\n

Literat\u00fara <\/strong><\/p>\n

    \n
  1. Woese CR. Bacterial evolution. Microbiol Rev 1987; 51(2): 221-271.<\/li>\n
  2. Rossello-Mora R, Amann R. Past and future species definitions for Bacteria and Archaea. Syst Appl Microbiol 2015; 38(4): 209-16.<\/li>\n
  3. Turcovsky I, Kunikova K, Drahovska H, Kaclikova E. Biochemical and molecular characterization of Cronobacter spp. (formerly Enterobacter sakazakii) isolated from foods. Antonie Van Leeuwenhoek 2011; 99(2): 257-69.<\/li>\n
  4. Sabat AJ. Overview of molecular typing methods for outbreak detection and epidemiological surveillance. Euro Surveill 2013; 18(4): 20380.<\/li>\n
  5. Urwin R, Maiden MCJ. Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol 2003; 11(10): 479-487.<\/li>\n
  6. Maiden MC, Jansen van Rensburg MJ, Bray JE, et al. MLST revisited: the gene-by-gene approach to bacterial genomics. Nat Rev Microbiol 2013; 11(10): 728-36.<\/li>\n
  7. Fleischmann RD, Adams MD, White O, et al. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 1995: 269(5223); 496.<\/li>\n
  8. Rossen JWA, Friedrich AW, Moran-Gilad J. Practical issues in implementing whole-genome-sequencing in routine diagnostic microbiology. Clin Microbiol Inf 2018; 24(4): 355-360.<\/li>\n
  9. Stasiewicz MJ, den Bakker HC, Wiedmann M. Genomics tools in microbial food safety. Current Opinion in Food Science 2015; 4: 105-110.<\/li>\n
  10. Larsen MV, Cosentino S, Lukjancenko O, et al. Benchmarking of Methods for Genomic Taxonomy. J Clin Microbiol 2014; 52: 1529.<\/li>\n
  11. Kadlicekova V, Kajsik M, Soltys K, et al. Characterisation of Cronobacter strains isolated from hospitalised adult patients. Antonie van Leeuwenhoek 2018; 111(7): 1073-1085.<\/li>\n
  12. Wattam AR, Davis JJ, Assaf R, et al. Improvements to PATRIC, the all-bacterial Bioinformatics Database and Analysis Resource Center. Nucleic Acids Res 2017; 45(D1): D535-D542.<\/li>\n
  13. Batchelor M, Hopkins KL, Liebana E, et al. Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria. Int. J. Antimicrob. Agents 2008; 31(5): 440-451.<\/li>\n
  14. Gupta SK, Padmanabhan BR, Diene SM, et al. ARG-annot, a new bioinformatic tool to discover antibiotic resistance genes in bacterial genomes. Antimicrob Agents Chemother 2014; 58(1): 212-220.<\/li>\n
  15. Zankari E, Hasman H, Cosentino S, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 2012; 67(11): 2640-4.<\/li>\n
  16. McArthur AG, Waglechner N, Nizam F, et al. The comprehensive antibiotic resistance database. Antimicrob Agents Chemother 2013; 57(7): 3348-57.<\/li>\n
  17. Ellington MJ, Ekelund O, Aarestrup FM, et al. The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the EUCAST Subcommittee. Clin Microbiol Infect 2017; 23(1): 2-22.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

    *All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf attachment at the end of the paper.   V klinickom mikrobiologickom laborat\u00f3riu sa na identifik\u00e1ciu a typiz\u00e1ciu p\u00f4vodcov infek\u010dn\u00fdch ochoren\u00ed tradi\u010dne pou\u017e\u00edvaj\u00fa fenotypov\u00e9 diagnostick\u00e9 met\u00f3dy zalo\u017een\u00e9 na kultiv\u00e1cii mikroorganizmov, mikroskopick\u00fdch technik\u00e1ch a \u0161pecializovan\u00fdch postupoch, ako je biotypiz\u00e1cia,<\/p>\n","protected":false},"author":7,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_mi_skip_tracking":false,"footnotes":""},"categories":[292],"tags":[1197,1196,1195,627],"class_list":["post-1729","post","type-post","status-publish","format-standard","hentry","category-microbiology","tag-antibiotic-resistance","tag-identification","tag-pathogen","tag-whole-genome-sequencing","typ_clanku-review-article"],"acf":{"abstrakt":"

    Presently, methods based on DNA sequencing are frequently used in clinical microbiology laboratory. Single locus sequencing is a traditional method used for bacterial identification and typing, 16S rRNA gene and MLST are the most frequently used ones. Recently the whole genome sequencing methods were applied in the clinical microbiology. This approach provides complex information about infectious agents. However, they demand advanced bioinformatic data evaluation. Bacterial genomes could be compared by several web-based applications; the examples are the whole genome MLST approach and PATRIC server. Several specialized databases are also devoted to detection of antibiotic resistance genes in sequenced bacterial genomes. <\/p>\n

    Keywords: pathogen, whole genome sequencing, identification, antibiotic resistance <\/p>\n","casopis":[{"ID":1633,"post_author":"7","post_date":"2019-05-09 08:56:48","post_date_gmt":"2019-05-09 06:56:48","post_content":"

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    • DNA sequencing in laboratory diagnostics of bacterial pathogens<\/li>\r\n \t
    • Importance and diagnostic of anti-phospholipid antibodies in women with reproductive disorders<\/li>\r\n \t
    • Development of complex chromosome rearrangements in a patient with ALL<\/li>\r\n \t
    • HCV infection \u2013 more than 20 years of drug development<\/li>\r\n<\/ul>","post_title":"newsLab","post_excerpt":"","post_status":"publish","comment_status":"closed","ping_status":"closed","post_password":"","post_name":"newslab-3","to_ping":"","pinged":"","post_modified":"2019-05-09 08:56:48","post_modified_gmt":"2019-05-09 06:56:48","post_content_filtered":"","post_parent":0,"guid":"http:\/\/www.newslab.sk\/?post_type=casopis&p=1633","menu_order":0,"post_type":"casopis","post_mime_type":"","comment_count":"0","filter":"raw"}],"strana":"25-27","upload_clanok":{"ID":1727,"id":1727,"title":"Sekvenovanie DNA v laborat\u00f3rnej diagnostike bakteri\u00e1lnych patog\u00e9nov","filename":"Sekvenovanie-DNA-v-laborat\u00f3rnej-diagnostike-bakteri\u00e1lnych-patog\u00e9nov.pdf","filesize":158681,"url":"https:\/\/www.newslab.sk\/wp-content\/uploads\/2019\/05\/Sekvenovanie-DNA-v-laborat\u00f3rnej-diagnostike-bakteri\u00e1lnych-patog\u00e9nov.pdf","link":"https:\/\/www.newslab.sk\/en\/dna-sequencing-in-laboratory-diagnostics-of-bacterial-pathogens\/sekvenovanie-dna-v-laboratornej-diagnostike-bakterialnych-patogenov-2\/","alt":"","author":"7","description":"","caption":"","name":"sekvenovanie-dna-v-laboratornej-diagnostike-bakterialnych-patogenov-2","status":"inherit","uploaded_to":1729,"date":"2019-05-09 08:59:57","modified":"2019-05-09 08:59:57","menu_order":0,"mime_type":"application\/pdf","type":"application","subtype":"pdf","icon":"https:\/\/www.newslab.sk\/wp-includes\/images\/media\/document.png"}},"_links":{"self":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1729","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/users\/7"}],"replies":[{"embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/comments?post=1729"}],"version-history":[{"count":0,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1729\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/media?parent=1729"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/categories?post=1729"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/tags?post=1729"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}