{"id":1008,"date":"2017-04-06T22:10:18","date_gmt":"2017-04-06T20:10:18","guid":{"rendered":"http:\/\/www.newslab.sk\/2017\/04\/06\/molekularno-geneticka-charakterizacia-koznych-bakterii\/"},"modified":"2017-10-03T08:09:38","modified_gmt":"2017-10-03T06:09:38","slug":"molecular-genetic-characteristics-of-skin-bacteria","status":"publish","type":"post","link":"https:\/\/www.newslab.sk\/en\/molecular-genetic-characteristics-of-skin-bacteria\/","title":{"rendered":"Molecular-genetic characteristics of skin bacteria"},"content":{"rendered":"<pre><span style=\"color: #ff0000;\"><strong>*All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf\u00a0\r\nattachment at the end of the paper.<\/strong><\/span><\/pre>\n<p>&nbsp;<\/p>\n<p><strong>\u00dav<\/strong><strong>od<\/strong><\/p>\n<p>Najv\u00e4\u010d\u0161\u00edm org\u00e1nom \u013eudsk\u00e9ho tela je ko\u017ea. \u00a0Okrem svojej prim\u00e1rnej funkcie (fyzick\u00e1 bari\u00e9ra medzi vonkaj\u0161\u00edm a vn\u00fatorn\u00fdm prostred\u00edm tela, \u00a0ochrana tela \u00a0pred \u00a0nebezpe\u010dn\u00fdmi l\u00e1tkami a cudz\u00edmi \u00a0\u0161kodliv\u00fdmi organizmami) je ko\u017ea domovom pre\u00a0 nespo\u010detn\u00e9 \u00a0mno\u017estvo mikroorganizmov. Najv\u00e4\u010d\u0161ie zast\u00fapenie maj\u00fa bakt\u00e9rie. Spolu \u00a0s hubami, v\u00edrusmi a mal\u00fdmi eukaryotick\u00fdmi organizmami, ako s\u00fa rozto\u010de, tvoria tzv. ko\u017en\u00fd mikrobi\u00f3m.<\/p>\n<p>Ko\u017en\u00e9 bakt\u00e9rie boli a s\u00fa \u0161tudovan\u00e9 ako potenci\u00e1lna pr\u00ed\u010dina vzniku r\u00f4znych chor\u00f4b. Dnes v\u0161ak vieme, \u017ee mnoh\u00e9 z nich nemaj\u00fa negat\u00edvny vplyv na zdravie \u00a0jedinca \u00a0\u010di dokonca pozit\u00edvne ovplyv\u0148uj\u00fa hostite\u013ea. Bolo preuk\u00e1zan\u00e9, \u017ee niektor\u00e9 bakt\u00e9rie pozit\u00edvne \u00a0ovplyv\u0148uj\u00fa imunitn\u00fd syst\u00e9m hostite\u013ea tak, \u017ee dok\u00e1\u017eu imunitn\u00fa odpove\u010f modifikova\u0165 \u00a0a dop\u013a\u0148a\u0165. Napr\u00edklad dok\u00e1\u017eu zvy\u0161ova\u0165 efekt\u00edvnos\u0165 T-buniek pri protiz\u00e1palovej imunitnej odpovedi(16).<\/p>\n<p>Ko\u017ea \u00a0funguje \u00a0ako \u00a0osobit\u00fd ekosyst\u00e9m. Nach\u00e1dza sa tu 1,8 m2\u00a0 r\u00f4znych \u00a0habitatov, z\u00e1hybov, invagin\u00e1ci\u00ed \u00a0a v\u00fdklenkov, ktor\u00e9 zabezpe\u010duj\u00fa nepravideln\u00fa koloniz\u00e1ciu ko\u017ee r\u00f4znymi bakteri\u00e1lnymi druhmi. \u00a0Zast\u00fapenie bakteri\u00e1lnych druhov\u00a0 z\u00e1vis\u00ed od toho, ak\u00e9 \u00a0s\u00fa\u00a0 podmienky v jednotliv\u00fdch \u00a0lokalit\u00e1ch na ko\u017ei. Rozli\u0161ujeme such\u00e9, vlhk\u00e9 a mastn\u00e9 miesta <strong><em>(obr\u00e1zok 1)<\/em><\/strong>. V oblastiach na ko\u017ei, ktor\u00e9 s\u00fa najviac vystaven\u00e9 vzduchu, prevl\u00e1da kme\u0148 <em>Proteobacteria<\/em>. Oblasti, kde sa nach\u00e1dza najviac mazov\u00fdch \u017eliaz, ako je chrb\u00e1t a tv\u00e1r, kolonizuj\u00fa najm\u00e4 bakt\u00e9rie z kme\u0148a <em>Actiniobacteria<\/em>. Miesta \u00a0s najvy\u0161\u0161ou vlhkos\u0165ou, ako je chodidlo, s\u00fa pokryt\u00e9 hlavne bakt\u00e9riami z kme\u0148a <em>Firmi<\/em><em>cutes. <\/em>Okrem t\u00fdchto \u00a0krit\u00e9ri\u00ed sa mno\u017estvo a rozlo\u017eenie bakt\u00e9ri\u00ed menia aj po\u010das rastu a dospievania jedinca, v z\u00e1vislosti od \u017eivotospr\u00e1vy a miesta \u017eivota(2,9,10).<\/p>\n<p>Rovnov\u00e1ha medzi \u00a0\u013eudsk\u00fdmi ko\u017en\u00fdmi bunkami a ko\u017en\u00fdmi bakt\u00e9riami je ve\u013emi d\u00f4le\u017eit\u00e1. Taktie\u017e je nevyhnut\u00e9, aby sa zachovala rovnov\u00e1ha medzi \u00a0bakteri\u00e1lnymi druhmi \u00a0navz\u00e1jom. Tzv. spont\u00e1nna necielen\u00e1 dysbalancia m\u00f4\u017ee \u00a0vies\u0165 a\u017e ku ko\u017en\u00fdm ochoreniam. Necielenou dysbalanciou rozumieme zmenu v zast\u00fapen\u00ed ko\u017en\u00fdch bakt\u00e9ri\u00ed \u00a0na ur\u010ditom mieste na tele. T\u00e1to \u00a0zmena m\u00f4\u017ee by\u0165 vyvolan\u00e1 \u00a0vystaven\u00edm ko\u017ee nevhodn\u00fdm podmienkam, nevhodn\u00e1 \u017eivotospr\u00e1va alebo dysbalancia m\u00f4\u017ee \u00a0by\u0165 geneticky zak\u00f3dovan\u00e1. Vedci sa sna\u017eia odhali\u0165 dysbalanciu detailne a ak je to mo\u017en\u00e9, \u00a0spoji\u0165 ju s ur\u010dit\u00fdm ochoren\u00edm. Dnes \u00a0vieme, \u00a0\u017ee \u00a0napr\u00edklad ochorenie atopickej dermatit\u00eddy je spojen\u00e9 so zn\u00ed\u017een\u00edm v\u00fdskytu <em>Staphylococcus <\/em>a zn\u00ed\u017een\u00edm celkovej diverzity ko\u017en\u00fdch bakt\u00e9ri\u00ed. \u00a0Viac asoci\u00e1ci\u00ed ochorenia s ko\u017en\u00fdm mikrobi\u00f3mom uv\u00e1dza <strong><em>tabu\u013eka 1<\/em><\/strong>(6,9,12) Cielen\u00e1 zmena mikrobi\u00f3mu sa sna\u017e\u00ed vyu\u017ei\u0165 poznatky o po\u0161koden\u00ed rovnov\u00e1hy \u00a0na lie\u010denie ko\u017en\u00fdch \u00a0ochoren\u00ed. Ak budeme \u00a0vedie\u0165 presn\u00fa pr\u00ed\u010dinu ko\u017en\u00e9ho ochorenia a bude \u00a0sa to t\u00fdka\u0165 \u00a0dysbalancie\u00a0 medzi\u00a0 \u00a0ko\u017en\u00fdmi bunkami \u00a0a \u00a0bakt\u00e9riami na \u00a0ko\u017ei, budeme vedie\u0165 \u00a0navrhn\u00fa\u0165 najefekt\u00edvnej\u0161ie lie\u010denie\u00a0ochorenia. Pod cielenou zmenou mikrobi\u00f3mu sa skr\u00fdvaj\u00fa r\u00f4zne \u00a0pr\u00edstupy. \u00a0Jednou z mo\u017enost\u00ed je genetick\u00e1 modifik\u00e1cia ko\u017en\u00fdch bakt\u00e9ri\u00ed s cie\u013eom zv\u00fd\u0161i\u0165 tvorbu bakterioc\u00ednov, \u010do sa vykon\u00e1va \u00a0naj\u010dastej\u0161ie klonovan\u00edm do bakteri\u00e1lnych plazmidov z ko\u017en\u00fdch \u00a0bakt\u00e9ri\u00ed, ktor\u00e9\u00a0 sa nach\u00e1dzaj\u00fa na ko\u017ei pacienta. \u010eal\u0161ou \u00a0mo\u017enos\u0165ou je umel\u00e9 zv\u00fd\u0161enie po\u010dtu \u00a0ch\u00fdbaj\u00facich druhov bakt\u00e9ri\u00ed na ur\u010ditom mieste na ko\u017ei(10).<\/p>\n<p>Aby sme mohli pracova\u0165 s ko\u017en\u00fdm mikrobi\u00f3mom, mus\u00edme sa nau\u010di\u0165 pracova\u0165 \u00a0s ko\u017en\u00fdmi bakt\u00e9riami nach\u00e1dzaj\u00facimi sa na ko\u017ei pacienta. Ke\u010f\u017ee to nie s\u00fa laborat\u00f3rne kmene, \u00a0pr\u00e1ca s nimi nie je trivi\u00e1lna. Je nutn\u00e9 \u00a0n\u00e1js\u0165 najefekt\u00edvnej\u0161\u00ed postup pr\u00e1ce. V\u0161etky postupy od sp\u00f4sobu odberu cez sp\u00f4sob kultiv\u00e1cie, izol\u00e1cie genomickej a plazmidovej DNA, identifik\u00e1cie bakteri\u00e1lnych druhov \u00a0a vyh\u013ead\u00e1vania bakteri\u00e1lnych plazmi- dov a g\u00e9nov s\u00fa nevyhnut\u00e9 optimalizova\u0165.<\/p>\n<p><strong>Materi\u00e1l a metodika<\/strong><\/p>\n<p>Na pokus boli pou\u017eit\u00e9 stery z ko\u017ee \u010dloveka \u00a0(vn\u00fatorn\u00e1 strana predlaktia a lak\u0165ov\u00e1 jamka, 4 zdrav\u00ed \u013eudia, ka\u017ed\u00fd zo sp\u00f4sobu odberu bol vykonan\u00fd nasleduj\u00faci de\u0148, po\u017eiadavky na hygienu odberov\u00e9ho miesta boli rovnak\u00e9 \u00a0pre v\u0161etk\u00fdch \u2013 sprchova\u0165 sa nami dodan\u00fdm mydlom \u00a0maxim\u00e1lne 24 hod\u00edn pred odberom) a stery z ko\u017ee bezsrstej my\u0161i pomocou sterilnej vatovej ty\u010dinky Swab plastic stem (Sarstedt, Numbrecht, Germany). T\u00e1 sa nam\u00e1\u010dala do siedmich r\u00f4znych roztokov (MBGW (molecular biology grade water), NaCl (chlorid sodn\u00fd), Tween-20 (polysorbate 20), PBS (fosf\u00e1tov\u00fd pufer), SCF (colony stimulating factor), EtOH (etanol) a NaCl + Tween-20) a sledovali sa pr\u00edpadn\u00e9 rozdiely. Bakt\u00e9rie boli po odbere vysiate na tuh\u00e9 LB m\u00e9dium, \u00a0kde bolo mo\u017en\u00e9 separova\u0165 jednotliv\u00e9 bakteri\u00e1lne druhy. Na namno\u017eenie bakt\u00e9ri\u00ed bolo pou\u017eit\u00e9 tuh\u00e9 a tekut\u00e9 LB m\u00e9dium, pri\u010dom sa pozoroval v\u00fd\u0165a\u017eok. Boli zvolen\u00e9 tri pr\u00edstupy izol\u00e1cie bakteri\u00e1lnej genomickej DNA, ktor\u00fdch efekt\u00edvnos\u0165 bola porovn\u00e1van\u00e1. Bakteri\u00e1lne druhy sa identifikovali pomocou Sangerovho sek- venovania. Bakteri\u00e1lna plazmidov\u00e1 DNA bola izolovan\u00e1 pou\u017eit\u00edm dvoch izola\u010dn\u00fdch kitov, ktor\u00e9 boli porovn\u00e1van\u00e9 medzi \u00a0sebou. N\u00e1sledne sa plazmidy \u00a0identifikovali \u00a0pomocou cielen\u00fdch PCR esej\u00ed. Mno\u017estvo DNA sa v ka\u017edom z uveden\u00fdch pr\u00edpadov meralo pomocou pr\u00edstroja Qubit\u00ae \u00a02.0 Fluorometer.<\/p>\n<p>Na vyhodnotenie d\u00e1t pri porovn\u00e1van\u00ed v\u00fdsledkov bol vyu\u017eit\u00fd \u0161tatistick\u00fd nep\u00e1rov\u00fd one-tailed t-test, \u00a0ktor\u00fd porovn\u00e1va stredn\u00e9 hodnoty a je schopn\u00fd porovna\u0165 \u00a0dve vari\u00e1cie \u00a0a pri hladine v\u00fdznamnosti nami \u00a0zvolenej (5 %) ur\u010d\u00ed, \u010di je rozdiel \u0161tatisticky v\u00fdznamn\u00fd, alebo \u00a0nie je. Nep\u00e1rov\u00fd t-test \u00a0bol zvolen\u00fd z toho d\u00f4vodu, \u017ee jednotliv\u00e9 \u00a0nameran\u00e9 koncentr\u00e1cie sa navz\u00e1jom neovplyv\u0148uj\u00fa. \u00a0Bola zvolen\u00e1 \u00a0one-tailed anal\u00fdza, lebo distrib\u00facia koncentr\u00e1ci\u00ed bola iba v kladn\u00fdch hodnot\u00e1ch na osi X. Taktie\u017e treba \u00a0uvies\u0165, \u017ee v\u0161etky d\u00e1ta \u00a0v r\u00e1mci pr\u00e1ce boli testovan\u00e9 Shapirov\u00fdm-Wilkov\u00fdm \u00a0testom na to, aby sa potvrdilo, \u017ee poch\u00e1dzaj\u00fa z norm\u00e1lneho rozdelenia. V\u0161etky d\u00e1ta \u00a0mali charakter norm\u00e1lneho rozdelenia, tak\u017ee \u00a0bolo mo\u017en\u00e9 pou\u017e\u00edva\u0165 parametrick\u00e9 \u0161tatistick\u00e9 testy.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>V<\/strong><strong>\u00fdsledky<\/strong><\/p>\n<p>&nbsp;<\/p>\n<p><strong><em>Optimaliz\u00e1cia \u00a0sp\u00f4sobu odberu bakt\u00e9ri\u00ed z ko\u017ee<\/em><\/strong><\/p>\n<p>Bola porovn\u00e1van\u00e1 koncentr\u00e1cia DNA, ktor\u00e1 \u00a0bola \u00a0z\u00edskan\u00e1 pou\u017eit\u00edm \u00a0r\u00f4znych \u00a0druhov\u00a0 roztokov. \u00a0Do t\u00fdchto roztokov \u00a0bola nam\u00e1\u010dan\u00e1 vatov\u00e1 \u00a0ty\u010dinka. Nulov\u00e1 hypot\u00e9za bola stanoven\u00e1 tak, \u017ee typ roztoku \u00a0nem\u00e1 vplyv na mno\u017estvo z\u00edskanej bakteri\u00e1lnej DNA. V\u00fdsledky s\u00fa uveden\u00e9 <strong><em>na obr\u00e1zku 2 v\u013eavo<\/em><\/strong>.<\/p>\n<p>P-hodnota vz\u00e1jomn\u00e9ho porovn\u00e1vania r\u00f4znych \u00a0druhov\u00a0 roztokov sa pohybuje \u00a0ni\u017e\u0161ie \u00a0od hodnoty 0,05. V jednom pr\u00edpade dokonca pod \u00farov\u0148ou 0,01. Z v\u00fdsledku vypl\u00fdva, \u017ee roztok NaCl + Tween 20 nie je vhodn\u00fd na tento \u00a0typ laborat\u00f3rnej pr\u00e1ce.<\/p>\n<p>Medzi \u00a0ostatn\u00fdmi roztokmi sa nezistil \u00a0\u017eiadny \u00a0v\u00fdznamn\u00fd \u0161tatistick\u00fd rozdiel, \u00a0tak\u017ee v\u00fdber roztoku neovplyvn\u00ed v\u00fd\u0165a\u017eok bakteri\u00e1lnej genomickej DNA.<\/p>\n<p>&nbsp;<\/p>\n<p><strong><em>K<\/em><\/strong><strong><em>ultiv\u00e1cia ko\u017en\u00fdch \u00a0bakt\u00e9ri\u00ed<\/em><\/strong><\/p>\n<p>Testovalo sa, \u010di konzistencia LB m\u00e9dia \u00a0a pr\u00edtomnos\u0165\/nepr\u00edtomnos\u0165 agaru m\u00e1 vplyv na v\u00fd\u0165a\u017eok bakteri\u00e1lnej genomickej DNA. Bolo kultivovan\u00fdch 13 \u00a0nami z\u00edskan\u00fdch bakteri\u00e1lnych druhov, ktor\u00e9 bolo nutn\u00e9 \u00a0namno\u017ei\u0165.<\/p>\n<p>Nulov\u00e1 hypot\u00e9za bola stanoven\u00e1 tak, \u017ee typ kultiva\u010dn\u00e9ho m\u00e9dia \u00a0nem\u00e1 vplyv na v\u00fd\u0165a\u017eok DNA. V\u00fdsledky porovn\u00e1vania s\u00fa uveden\u00e9 <strong><em>na obr\u00e1zku 2 vpravo<\/em><\/strong>.<\/p>\n<p>P-hodnota je na \u00farovni 0,4917, \u010do znamen\u00e1 potvrdenie nulovej hypot\u00e9zy.<\/p>\n<p>&nbsp;<\/p>\n<p><strong><em>Izol\u00e1cia bakteri\u00e1lnej genomickej DNA<\/em><\/strong><\/p>\n<p>Experiment\u00e1lne boli porovn\u00e1van\u00e9 tri postupy izol\u00e1cie:<\/p>\n<ol>\n<li>Izola\u010dn\u00fd kit QIAamp\u00ae DNA Mini kit 250;<\/li>\n<li>Optimalizovan\u00fd protokol izola\u010dn\u00e9ho kitu QIAamp\u00ae DNA Mini kit 250 (doplnenie p\u00f4vodn\u00e9ho protokolu o teplotn\u00e9 \u0161oky pred prv\u00fdm krokom 5x striedanie 5 min 90 \u00baC\/5 min \u201320 \u00a0\u00baC);<\/li>\n<li>Optimalizovan\u00fd protokol (zmena oproti p\u00f4vodn\u00e9mu protokolu \u2013 pufor ATL bol nahraden\u00fd lyza\u010dn\u00fdm roztokom; bol pridan\u00fd roztok lyzoz\u00fdmu, roztok RN\u00e1zy; pred pridan\u00edm protein\u00e1zy K bola pridan\u00e1 hodinov\u00e1 inkub\u00e1cia pri 37 \u00baC, po pridan\u00ed \u00a0pufra \u00a0AL bola \u00a0pridan\u00e1 \u00a030-min\u00fatov\u00e1 \u00a0inkub\u00e1cia \u00a0pri 55 \u00baC a n\u00e1sledne 30-min\u00fatov\u00e1 inkub\u00e1cia pri 70 \u00baC).Vz\u00e1jomn\u00e9 porovn\u00e1vanie v\u00fdsledkov demon\u0161truje <strong><em>obr\u00e1zok \u00a03 v\u013eavo<\/em><\/strong>. Nulov\u00e1 hypot\u00e9za bola stanoven\u00e1 tak, \u017ee typ protokolu nem\u00e1 vplyv na mno\u017estvo izolovanej DNA. V\u00fdsledkom porovnania p\u00f4vodn\u00e9ho protokolu s 1. optimalizovan\u00fdm protokolom je P-hodnota na \u00farovni 0,3388. \u00a0Porovnanie p\u00f4vodn\u00e9ho protokolu s 2. optimalizovan\u00fdm protokolom d\u00e1va P-hodnotu na \u00farovni 0,0150 \u00a0a porovnanie 1. optimalizovan\u00e9ho protokolu s 2. optimalizovan\u00fdm protokolom d\u00e1va \u00a0P-hodnotu 0,0178. \u00a0Mo\u017eno teda poveda\u0165, \u017ee nulov\u00e1 hypot\u00e9za sa zamieta v prospech 2. optimalizovan\u00e9ho protokolu, s ktor\u00fdm sme boli schopn\u00ed izolova\u0165 najv\u00e4\u010d\u0161iu koncentr\u00e1ciu bakteri\u00e1lnej genomickej DNA.<strong><em>Identifik\u00e1cia z\u00edskan\u00fdch bakteri\u00e1lnych\u00a0 druhov pomocou<\/em><\/strong><strong><em>Sangerovho <\/em><\/strong><strong><em>sekv<\/em><\/strong><strong><em>eno<\/em><\/strong><strong><em>v<\/em><\/strong><strong><em>ania<\/em><\/strong>Bakteri\u00e1lne druhy boli identifikovan\u00e9 pomocou klasickej met\u00f3dy Sangerovho sekvenovania 16S \u00a0rDNA. Podarilo \u00a0sa n\u00e1m \u00a0z\u00edska\u0165 12 r\u00f4znych bakteri\u00e1lnych druhov \u2013 7 zo sterov z ko\u017ee \u010dloveka \u00a0a 7 z ko\u017ee my\u0161i, pri\u010dom \u00a0dva druhy sa vyskytli aj u \u010dloveka \u00a0aj u my\u0161i. Jednotliv\u00e9 druhy s\u00fa\u00a0 uveden\u00e9 v <strong><em>tabu\u013eke 2<\/em><\/strong>.&nbsp;\n<p><strong><em>Izol\u00e1cia bakteri\u00e1lnej plazmidovej DNA<\/em><\/strong><\/p>\n<p>Zo v\u0161etk\u00fdch nami \u00a0z\u00edskan\u00fdch bakteri\u00e1lnych druhov \u00a0bolo vybrat\u00fdch \u00a0\u0161es\u0165, z ktor\u00fdch \u00a0sa izolovala plazmidov\u00e1 DNA. Boli porovnan\u00e9 dva izola\u010dn\u00e9 kity DNA QIAprep Spin Miniprep kit a NucleoBond\u00ae Xtra Midi. V\u00fdsledky s\u00fa demon\u0161trovan\u00e9 <strong><em>na obr\u00e1zku 3 vpravo<\/em><\/strong>.<\/p>\n<p>Nulov\u00e1 hypot\u00e9za bola tradi\u010dne stanoven\u00e1 tak, \u017ee typ izola\u010dn\u00e9ho kitu nem\u00e1 vplyv na mno\u017estvo izolovanej plazmidovej DNA. Hypot\u00e9za v\u0161ak bola vzh\u013eadom na v\u00fdsledky zamietnut\u00e1 v prospech kitu NucleoBond\u00ae Xtra Midi.<\/p>\n<p><strong><em>Identifik\u00e1cia pr\u00edtomn\u00fdch \u00a0plazmidov<\/em><\/strong><\/p>\n<p>Jedn\u00fdm zo sp\u00f4sobov, ako vyh\u013eada\u0165 a identifikova\u0165 pr\u00edtomn\u00e9 plazmidy, je pou\u017eitie \u00a0cielen\u00fdch PCR esej\u00ed. Na tento \u00a0experiment \u00a0bol vybran\u00fd bakteri\u00e1lny druh S<em>taphylococcus <\/em><em>epider<\/em><em>midis<\/em>, v ktorom \u00a0sa vyh\u013ead\u00e1vali potenci\u00e1lne pr\u00edtomn\u00e9 vlastn\u00e9 plazmidy. Bolo navrhnut\u00fdch \u0161es\u0165 p\u00e1rov primerov na \u0161es\u0165 r\u00f4znych plazmidov. N\u00e1sledne bolo v\u0161etk\u00fdch \u0161es\u0165 potenci\u00e1lnych PCR produktov nanesen\u00fdch na agar\u00f3zov\u00fd g\u00e9l <strong><em>(obr\u00e1zok \u00a04).\u00a0<\/em><\/strong>Cielen\u00fdmi PCR esejami bolo mo\u017en\u00e9 identifikova\u0165 dva plazmidy pr\u00edtomn\u00e9 v l\u00ednii nami z\u00edskanej bakt\u00e9rie <em>Staphylococcus <\/em><em>epidermidis<\/em>.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Diskusia<\/strong><\/p>\n<p>Pr\u00e1ca s bakt\u00e9riami, ktor\u00e9 nie s\u00fa klasick\u00fdmi laborat\u00f3rnymi kme\u0148mi, je ve\u013emi n\u00e1ro\u010dn\u00e9. Ka\u017ed\u00fd krok si vy\u017eaduje pr\u00e1cne optimalizovanie. Av\u0161ak bez toho by sme sa v tejto oblasti nemohli posun\u00fa\u0165 \u010falej, preto m\u00e1 toto \u00fasilie ve\u013ek\u00fd v\u00fdznam. V mojej pr\u00e1ci s\u00fa sumarizovan\u00e9 najvhodnej\u0161ie postupy molekul\u00e1rnogenetickej anal\u00fdzy nepatog\u00e9nnych nami z\u00edskan\u00fdch bakt\u00e9ri\u00ed z ko\u017ee. Bolo mo\u017en\u00e9 identifikova\u0165 najlep\u0161\u00ed postup pr\u00e1ce.<\/p>\n<p>Optimaliz\u00e1cia sp\u00f4sobu odberu ko\u017en\u00fdch \u00a0bakt\u00e9ri\u00ed \u00a0bola v\u00fdchodiskov\u00e1 \u00faloha pre \u010fal\u0161ie experimenty. Bolo vybrat\u00fdch sedem \u00a0roztokov, \u00a0do \u00a0ktor\u00fdch \u00a0sa nam\u00e1\u010dala vatov\u00e1 \u00a0ty\u010dinka, \u00a0na zv\u00fd\u0161enie v\u00fd\u0165a\u017eku bakteri\u00e1lnych buniek. Ka\u017ed\u00e1 z l\u00e1tok m\u00e1 potenci\u00e1l \u00a0pozit\u00edvne \u00a0ovplyvni\u0165 odber \u00a0bakt\u00e9ri\u00ed. \u00a0V\u00fdsledky testovania vyl\u00fa\u010dili roztok NaCl + Tween 20, preto\u017ee sa preuk\u00e1zal ako nevhodn\u00fd. D\u00f4vodom \u00a0m\u00f4\u017ee \u00a0by\u0165 rozdielna pH hodnota. Zatia\u013e \u010do pH hodnota ko\u017ee na predlakt\u00ed \u00a0po 24 hodin\u00e1ch po sprchovan\u00ed \u00a0a \u00a0nepou\u017eit\u00ed \u00a0\u017eiadnych kozmetick\u00fdch pr\u00edpravkov \u00a0je od 5,12 \u00b1 0,56 do 4,93 \u00b1 0,45(11), tak pH hodnota roztoku \u00a0je 7,6. Medzi ostatn\u00fdmi l\u00e1tkami nebol \u0161tatisticky v\u00fdznamn\u00fd rozdiel. Na z\u00e1ver v\u0161ak bolo mo\u017en\u00e9 ur\u010di\u0165 MBGW ako najide\u00e1lnej\u0161iu l\u00e1tku, lebo s jej pou\u017eit\u00edm sme z\u00edskali najmen\u0161iu odch\u00fdlku v meran\u00ed, m\u00e1 chemick\u00e9 zlo\u017eenie bez najmen\u0161\u00edch odch\u00fdlok v r\u00e1mci r\u00f4znych laborat\u00f3ri\u00ed, \u00a0\u010d\u00edm sa zabezpe\u010d\u00ed maxim\u00e1lna reprodukovate\u013enos\u0165 experimentu.\u017divn\u00e9 m\u00e9di\u00e1, \u00a0ktor\u00e9 \u00a0sa pou\u017e\u00edvaj\u00fa \u00a0na kultiv\u00e1ciu bakt\u00e9ri\u00ed, sa l\u00ed\u0161ia svoj\u00edm zlo\u017een\u00edm a konzistenciou. Ak by sme potrebovali z\u00edska\u0165 \u010do najviac bakteri\u00e1lnych druhov, ur\u010dite by sme zvolili r\u00f4zne \u00a0p\u00f4dy, experimentovalo by sa so zlo\u017ekami a testovali by sa selek\u010dn\u00e9 m\u00e9di\u00e1. V na\u0161om pr\u00edpade bola zvolen\u00e1 klasick\u00e1 LB p\u00f4da, \u00a0pomocou ktorej bolo mo\u017en\u00e9 z\u00edska\u0165 dostato\u010dn\u00e9 mno\u017estvo bakteri\u00e1lnych druhov. Av\u0161ak experiment\u00e1lne bolo zis\u0165ovan\u00e9, \u00a0\u010di konzistencia kultiva\u010dn\u00e9ho m\u00e9dia vpl\u00fdva na v\u00fd\u0165a\u017eok bakteri\u00e1lnej genomickej DNA. Nena\u0161li sa \u017eiadne signifikantn\u00e9 \u00a0rozdiely medzi \u00a0t\u00fdmito dvoma pr\u00edstupmi, preto mo\u017eno poveda\u0165, \u00a0\u017ee konzistencia m\u00e9dia nem\u00e1 vplyv na\u00a0 v\u00fd\u0165a\u017eok bakteri\u00e1lnej DNA.<\/p>\n<p>\u010eal\u0161\u00edm krokom anal\u00fdzy bola izol\u00e1cia bakteri\u00e1lnej genomickej \u00a0DNA na \u00a0n\u00e1sledn\u00e9 ur\u010denie z\u00edskan\u00fdch bakteri\u00e1lnych druhov. Na trhu s\u00fa dostupn\u00e9 r\u00f4zne izola\u010dn\u00e9 kity, ktor\u00e9 sa l\u00ed\u0161ia svojou \u00a0\u0161pecifickos\u0165ou na ur\u010dit\u00e9 \u00a0bakteri\u00e1lne druhy, \u010di schopnos\u0165ou \u00a0izol\u00e1cie grampozit\u00edvnych a gramnegat\u00edvnych bakt\u00e9ri\u00ed. Na na\u0161om pracovisku sa dlhodobo osved\u010dil izola\u010dn\u00fd \u00a0kit QIAamp\u00ae DNA Mini kit 250 od firmy Qiagen, preto bol zvolen\u00fd ako v\u00fdchodiskov\u00fd, ktor\u00fd bol n\u00e1sledne podroben\u00fd nieko\u013ek\u00fdm zmen\u00e1m. Z v\u00fdsledkov \u00a0vypl\u00fdva, \u017ee optimaliz\u00e1cie viedli k zv\u00fd\u0161eniu v\u00fd\u0165a\u017eku bakteri\u00e1lnej DNA, preto boli uveden\u00e9 do praxe.<\/p>\n<p>Bakteri\u00e1lne druhy boli identifikovan\u00e9 pomocou sekvenovania \u00a016S rDNA k\u00f3duj\u00facej sekvencie. T\u00e1to sekvencia obsahuje vysokokonzervovan\u00e9 \u00faseky \u00a0s vysokou \u00a0medzidruhovou variabilitou, \u010do je d\u00f4vodom, pre\u010do \u00a0je t\u00e1to \u00a0sekvencia vhodn\u00e1 pri identifik\u00e1cii bateri\u00e1lnych druhov(14,15). Na tento konzervovan\u00fd \u00fasek\u00a0 bolo navrhnut\u00fdch nieko\u013eko p\u00e1rov(13), z ktor\u00fdch boli pre t\u00fato \u0161t\u00fadiu\u00a0 vybrat\u00e9 dva \u2013 27F (5\u2019-AGAGTTTGATCCTGGC- TCAG-3\u2019) a 1492R (5\u2019-GGTTACCTTGTTACGACTT-3\u2019). V\u00fdsledkom bola \u00faspe\u0161n\u00e1 identifik\u00e1cia trin\u00e1stich bakteri\u00e1lnych druhov, ktor\u00e9 \u00a0s\u00fa \u00a0uveden\u00e9 v <strong><em>tabu\u013eke 2<\/em><\/strong>. Z nich bolo \u00a0na\u00a0 z\u00e1klade ur\u010den\u00fdch vlastnost\u00ed (nepatogenita a v\u00fdskyt na\u00a0 ko\u017ei) vybrat\u00fdch \u0161es\u0165, z ktor\u00fdch bola izolovan\u00e1 plazmidov\u00e1 DNA. Z ve\u013ekej \u0161k\u00e1ly dostupn\u00fdch izola\u010dn\u00fdch kitov boli vybrat\u00e9 dva kity, ktor\u00e9 boli porovn\u00e1van\u00e9. Na z\u00e1klade v\u00fdsledkov sa ako lep\u0161\u00ed uk\u00e1zal NucleoBond\u00ae Xtra Midi.<\/p>\n<p>Cielen\u00e1 \u00a0PCR esej je dobr\u00fdm \u00a0n\u00e1strojom na\u00a0 vyh\u013ead\u00e1vanie \u0161pecifick\u00fdch \u00fasekov DNA vo vzorke. Pr\u00e1ve t\u00fdmto\u00a0 pr\u00edstupom boli vyh\u013ead\u00e1van\u00e9 bakteri\u00e1lne plazmidy v na\u0161om experimente. Zo v\u0161etk\u00fdch z\u00edskan\u00fdch druhov \u00a0bol vybrat\u00fd iba <em>Staphylo<\/em><em>coccus <\/em><em>e<\/em><em>pi<\/em><em>d<\/em><em>e<\/em><em>r<\/em><em>m<\/em><em>i<\/em><em>d<\/em><em>i<\/em><em>s<\/em> preto, \u00a0\u017ee z druhov \u00a0nami \u00a0z\u00edskan\u00fdch m\u00e1 najviac op\u00edsan\u00fdch vlastn\u00fdch plazmidov so \u00a0zn\u00e1mou sekvenciou. \u00a0Navrhli sme \u0161es\u0165 p\u00e1rov \u00a0primerov \u00a0na \u00a0\u0161es\u0165 plazmidov. PCR produkty boli nanesen\u00e9 na g\u00e9lov\u00fa elektrofor\u00e9zu, na ktorej bola \u00faspe\u0161ne identifikovan\u00e1 pr\u00edtomnos\u0165 pr\u00e1ve dvoch bakteri\u00e1lnych plazmidov pUR3036 a pSK103.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Z\u00e1ver<\/strong><\/p>\n<p>Na to, aby bolo mo\u017en\u00e9 uva\u017eova\u0165 \u00a0o cielenej \u00a0zmene ko\u017en\u00e9ho mikrobi\u00f3mu, \u00a0je potrebn\u00e9 najprv sa nau\u010di\u0165 s nelaborat\u00f3rnymi bakt\u00e9riami pracova\u0165 \u00a0\u010do najlep\u0161ie a najefekt\u00edvnej\u0161ie. Na to n\u00e1m sl\u00fa\u017eia \u00a0pr\u00e1ve tak\u00e9to experimenty, ktor\u00e9 vymedzuj\u00fa tie najlep\u0161ie met\u00f3dy a postupy.<\/p>\n<p>Plazmidy, \u00a0ktor\u00e9 \u00a0boli \u00a0t\u00fdmto \u00a0postupom \u00a0z\u00edskan\u00e9, by mali by\u0165 n\u00e1sledne analyzovan\u00e9 a nesk\u00f4r potenci\u00e1lne vyu\u017eit\u00e9 pri alternat\u00edvnej g\u00e9novej \u00a0terapii. \u00a0G\u00e9n z\u00e1ujmu \u00a0by sa mohol \u00a0vklada\u0165 do t\u00fdch plazmidov, ktor\u00e9 by boli n\u00e1sledne vlo\u017een\u00e9 sp\u00e4\u0165 do bakt\u00e9rie. Takto pozmenen\u00e1 bakt\u00e9ria by sa mala \u00a0\u010falej analyzova\u0165 a \u0161tudova\u0165 jej potenci\u00e1lne op\u00e4tovn\u00e9 os\u00eddlenie hostite\u013esk\u00e9ho organizmu s cie\u013eom zdravotn\u00e9ho benefitu pre \u010dloveka pri lie\u010den\u00ed ko\u017en\u00fdch \u00a0ochoren\u00ed.<\/p>\n<p><strong>Literat\u00fara<\/strong><br \/>\n1. Alekseyenko AV, Perez-Perez GI, De Souza A. Community differentiation of the cutaneous microbiota in psoriasis. Microbiome 2013; 1(1): 31.<br \/>\n2. Baviera G, Leoni MCH, Capra L, Microbiota in healthy skin and in Atopic eczema. Hindawau Publishing Corporation, BioMed Research International 2014. ID 436921.<br \/>\n3. Casas C, Paul C, Lahfa M, et al. Quantification of Demodex folliculorum<br \/>\nby PCR in rosacea and its relationship to skin innate immune activation. Exp Dermatol 2012; 21(12): 906-10.<br \/>\n4. Dawson TL. Malassezia globosa and restricta: Breakthrough understanding of the etiology and treatment of dandruff and seborrheic dermatitis through whole-Genome analysis. J Invest Derm Symp 2007; P12: 15-19.<br \/>\n5. Fahlen A, Engstrand L, Baker BS, et al. Comparison of bacterial microbiota in skin biopsies from normal and psoriatic skin. Arch Dermatol Res 2012; 304(1): 15-22.<br \/>\n6. Findley K, Grice EA. The Skin Microbiome: A focus on pathogens and their association with skin disease. PLoS Pathogens 2014; 10(11): 1004436.<br \/>\n7. Fitz-Gibbon S, Tomida S, Chiu BH. Propionibacterium acnes strain populations in the human skin microbiome associated with acne. J Invest Dermatol 2013; 133(9): 2152-2160.<br \/>\n8. Gao Z, Tseng CH, Strober BE, et al. Substantial alterations of the cutaneous bacterial biota in psoriatic lesions. PLoS One 2008; 3(7): 2719.<br \/>\n9. Grice EA. The skin microbiome: potential for novel diagnostic and therapeutic approaches to cutaneous disease. Seminars in Cutaneous Medicine and surgery 2014; vol 33.<br \/>\n10. Kong HH, Oh J, Deming C, et al. Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis. Genome Res 2012; 22(5): 850-859.<br \/>\n11. Lambers H, Piessens S, Bloem A, Pronk H, Finkel P. Natural skin surface pH is on average below 5, which is beneficial for its resident flora. Int J Cosmet Sci 2006; 28(5): 359-70.<br \/>\n12. Tomida S, Nguyen L, Chiu B, et al. Pan-genome and comparative genome analyses of Propionibacterium acnes reveal its genomic diversity in the healthy and diseased human skin microbiome. MBio 2013; 4(3): 3-13.<br \/>\n13. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 1991; 173(2): 697-703.<br \/>\n14. Woese CR, Fox GE. Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proceedings of the National Academy of Sciences of the United States of America 1977; 74(11): 5088-5090.<br \/>\n15. Woo PCY, Lau SKP, et al. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Journal Compilation 2008; 14(10): 908-934.<br \/>\n16. Zeeuwen P, Boekhorst J, van den Bogaard EH, Bacterial pathogenesis. Microbiome dynamics of human epidermis following skin barrier disruption 2012. kap.7.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"<p>*All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf\u00a0 attachment at the end of the paper. &nbsp; \u00davod Najv\u00e4\u010d\u0161\u00edm org\u00e1nom \u013eudsk\u00e9ho tela je ko\u017ea. \u00a0Okrem svojej prim\u00e1rnej funkcie (fyzick\u00e1 bari\u00e9ra medzi vonkaj\u0161\u00edm a vn\u00fatorn\u00fdm prostred\u00edm tela, \u00a0ochrana tela \u00a0pred \u00a0nebezpe\u010dn\u00fdmi l\u00e1tkami a cudz\u00edmi \u00a0\u0161kodliv\u00fdmi organizmami) je<\/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":[298],"tags":[599,600,598,597],"class_list":["post-1008","post","type-post","status-publish","format-standard","hentry","category-molecular-biology","tag-cultivation-of-non-model-bacterial-species","tag-dna-isolation","tag-molecular-genetic-bacterial-analysis","tag-skin-microbiome","typ_clanku-original-work"],"acf":{"abstrakt":"<p>Skin bacteria have a demonstrable impact on health status of the host. With their genes, all human skin bacteria constitute a unique extension of the human genome. Thanks to detailed analyses of positive and negative association between the skin bacteria and health of their host, background of some skin diseases that are related to skin bacteria to a smaller or larger extent can be revealed.\u00a0 This knowledge can be potentially used in prevention and treatment of skin diseases by way of alternative targeted gene therapy. To achieve this goal, it is necessary to perform a complete molecular-genetic analysis of non-pathogenic skin bacteria and prove the link between them and the health of the host. However, these species of bacteria are not standard laboratory species and it is therefore necessary to optimize all procedures and analyses.<\/p>\n<p><strong>Key words:<\/strong> skin microbiome, molecular-genetic bacterial analysis, cultivation of non-model bacterial species, DNA isolation<\/p>\n","casopis":[{"ID":735,"post_author":"7","post_date":"2017-04-06 13:21:01","post_date_gmt":"2017-04-06 11:21:01","post_content":"<ul>\r\n \t<li>Pseudoglandular nevus \u2013 a rare morphology of melanocytic nevus (case report)<\/li>\r\n \t<li>Differential molecular diagnosis of multiple myeloma and Waldenstr\u00f6m macroglobulinemia<\/li>\r\n \t<li>Molecular analysis of prognostically significant markers of chronic lymphocytic leukemia<\/li>\r\n \t<li>Prevalence of <em>Streptococcus pneumoniae<\/em> phyla in inflammatory diseases of upper airways in preschool age children and their resistance to antibiotics<\/li>\r\n \t<li>Malign melanoma - new aspects of research<\/li>\r\n \t<li style=\"list-style-type: none;\"><\/li>\r\n<\/ul>","post_title":"Newslab","post_excerpt":"","post_status":"publish","comment_status":"closed","ping_status":"closed","post_password":"","post_name":"newslab-2017-1","to_ping":"","pinged":"","post_modified":"2017-08-16 21:11:52","post_modified_gmt":"2017-08-16 19:11:52","post_content_filtered":"","post_parent":0,"guid":"http:\/\/www.newslab.sk\/?post_type=casopis&#038;p=735\/","menu_order":0,"post_type":"casopis","post_mime_type":"","comment_count":"0","filter":"raw"}],"strana":"16","upload_clanok":{"ID":832,"id":832,"title":"Molekul\u00e1rno-genetick\u00e1 charakteriz\u00e1cia ko\u017en\u00fdch bakt\u00e9ri\u00ed","filename":"Molekul\u00e1rno-genetick\u00e1-charakteriz\u00e1cia-ko\u017en\u00fdch-bakt\u00e9ri\u00ed.pdf","filesize":883321,"url":"https:\/\/www.newslab.sk\/wp-content\/uploads\/2017\/04\/Molekul\u00e1rno-genetick\u00e1-charakteriz\u00e1cia-ko\u017en\u00fdch-bakt\u00e9ri\u00ed.pdf","link":"https:\/\/www.newslab.sk\/en\/molecular-genetic-characteristics-of-skin-bacteria\/molekularno-geneticka-charakterizacia-koznych-bakterii\/","alt":"","author":"7","description":"","caption":"","name":"molekularno-geneticka-charakterizacia-koznych-bakterii","status":"inherit","uploaded_to":1008,"date":"2017-04-06 19:46:23","modified":"2017-04-06 19:46:23","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\/1008","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=1008"}],"version-history":[{"count":0,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1008\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/media?parent=1008"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/categories?post=1008"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/tags?post=1008"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}