Molecular methods for monitoring contamination of hospital textiles

Bolnišnično okolje predstavlja pomembno ekološko nišo in lahko služi kot rezervoar za potencialno patogene mikroorganizme. Bolnišnične tekstilije skupaj z vlago in toploto ustvarijo ustrezne pogoje za rast, širjenje in dolgotrajno preživetje številnih mikroorganizmov, zaradi česar lahko služijo kot vektor navzkrižnega prenosa bolnišničnih okužb. Bolnišnične okužbe ne predstavljajo samo zapletov pri zdravljenju bolnikov v bolnišnici, zaradi katerih letno v Evropi umre 50.000 ljudi, temveč povzročajo tudi gospodarsko škodo, saj so letne finančne izgube v Evropi samo z direktnimi stroški ocenjene na približno 7 milijard EUR. Klasične metode za vzorčenje mikroorganizmov na tekstilijah, kot je jemanje odtisov z RODAC agar ploščami, brisi in z destruktivno elucijsko metodo ter naknadna fenotipska identifikacija, so dolgotrajne, zato smo v raziskavi uvedli vzorčenje z metodo nedestruktivnega eluiranja z aparaturo Morapex A in detekcijo mikroorganizmov z verižno reakcijo s polimerazo (PCR) ter kvantifikacijo z verižno reakcijo s polimerazo v realnem času (rtPCR). Dokazali smo, da aparatura Morapex A predstavlja ustrezen in učinkovit nadomestek za vzorčenje mikroorganizmov na tekstilijah, saj je bila učinkovitost vzorčenja večja v primerjavi z optimizirano metodo destruktivnega eluiranja oz. z metodo vzorčenja z RODAC agar ploščami. Z uporabo molekularnih metod za detekcijo izbranih vrst mikroorganizmov smo dosegli večjo občutljivost kot pri detekciji s klasičnimi gojitvenimi metodami, saj lahko mikroorganizme zaznamo pri nižjih začetnih nanesenih koncentracijah na tekstil, zaznamo pa tudi žive in mrtve mikroorganizme ter mikroorganizme v VBNC stanju in ostanke proste DNK. Za oceno celotne mikrobne populacije na tekstilijah iz realnega okolja smo uporabili metodo ločevanja celotne bakterijske 16S rDNK z visokotlačno kromatografijo v denaturizirajočih pogojih (DHPLC). Zaradi široke pestrosti mikrobne populacije na bolnišničnih tekstilijah iz realnega okolja se metoda DHPLC ni izkazala kot najbolj ustrezna, saj zaradi pojavljanja mešanih sekvenc v posamezni frakciji ni bila mogoča identifikacijo do ene same bakterijske vrste. Vzorčenje mikroorganizmov na tekstilijah z metodo nedestruktivnega eluiranja z aparaturo Morapex A in njihova detekcija z molekularnimi metodami lahko predstavlja izboljšano alternativo za potrebe kontrole higiene bolnišničnih tekstilij.Hospital environment provides an important ecological niche that can serve as a reservoir for potentially pathogenic microorganisms. Hospital textiles together with moisture and heat create the right conditions for growth, dissemination, and long-term survival of many micro-organisms, which may serve as a vector for cross-transmission of nosocomial infections. Nosocomial infections represent not only complications in the treatment of patients in hospital that annually kill 50,000 people, but also cause economic damage with estimated annual financial losses at approximately 7 billion EUR only for direct costs. Classical methods for sampling microorganisms on textiles, using RODAC agar plates, swabs, and destructive elution method followed by phenotypic identification are time consuming. In this study, we have introduced the nondestructive elution sampling method using the Morapex A device followed by identification of microorganisms with polymerase chain reaction (PCR) and their quantitation with real time polymerase chain reaction (rtPCR). It has been proven that the Morapex A device represents an adequate and effective substitute for sampling microorganisms on fabrics since the method efficiency has been higher when compared to the optimized destructive elution method or to sampling method with RODAC agar plates. With the use of molecular methods for the identification of selected species of micro-organisms, the sensitivity has been higher than identification with traditional cultivation methods, since microorganisms can be detected at lower initially applied concentration of microorganisms on textiles. Also live and dead microorganisms, microorganisms in the VBNC state, and remains of free DNA have been detected. To assess the overall microbial populations in textiles from the real environment, the separation of 16S rDNA with DHPLC method has been used. Due to the wide diversity of microbial populations on the hospital textiles from the real environment the method DHPLC has not been the most appropriate because it has not been possible to identify a single bacterial species due to the emergence of mixed sequences in each fraction. Sampling of microorganisms on textiles with destructive elution method using Morapex A and their identification with molecular methods may represent an improved alternative for the verification of the hygiene of hospital textiles