Kolonizacija vankomicin-rezistentnim Enterococcus spp. sojevima u bolničkoj sredini - genotipska i fenotipska karakterizacija sojeva i faktori rizika za kolonizaciju

Abstract

Detekcija fekalne kolonizacije vankomicin-rezistentnim Enterococcus spp. (VRE) sojevima ubolničkoj sredini ima veliki značaj u nadzoru nad bolničkom infekcijom s obzirom da je poznato da VREkolonizacija najčešće prethodi VRE infekciji. Ovo istraživanje predstavlja prvo epidemiološkomikrobiološkoistraživanje o fekalnoj kolonizaciji VRE sojevima kod hospitalizovanih pacijenata naodelјenjima sa povišenim rizikom za nastanak VRE kolonizacije.CILJEVI: Ciljevi ovog istraživanja su bili: (1) utvrđivanje učestalosti fekalne kolonizacije VREsojevima kod hospitalizovanih pacijenata na odelјenjima sa povišenim rizikom za nastanak kolonizacije;(2) identifikacija faktora rizika za fekalnu VRE kolonizaciju; (3) ispitivanje fenotipskih i genotipskihkarakteristika izolovanih VRE sojeva i utvrđivanje klonalne povezanosti i klonalne diseminacije VREsojeva.MATERIJAL I METODE: Istraživanje je uključilo 268 ispitanika sa šest odeljenja u tri univerzitetskebolnice u Beogradu. Za ispitivanje faktora rizika (FR) korišćena je multivarijantna logistička regresija.Karakterizacija VRE izolata obuhvatila je određivanje profila rezistencije primenom automatizovanogBD Phoenix™ sistema, molekularnu identifikaciju detekcijom vrsno specifičnih gena (ddlE. faecium, ddlE.faecalis), detekciju gena rezistencije na glikopeptidne antimikrobne lekove (vanA, vanB, vanC1, vanC2/C3), detekciju gena rezistencije na kvinupristin-dalfopristin (Q-D) (vatD, vatE, vgbA, ermB1),detekciju gena za faktore virulencije (esp, hyl, efaA, asa1, gelE, cpd) i MLVA analizu (eng. MultiplelocusVariable-number tandem repeat analysis). Ispitivanje sposobnosti formiranja biofilma rađeno jemetodom u mikrotitracionoj ploči.REZULTATI: Učestalost fekalne VRE kolonizacije je iznosila 28,7%. Nezavisni prediktori za nastanakVRE kolonizacije među ispitanicima hospitalizovanim na kliničkim odeljenjima sa povišenim rizikomza nastanak VRE kolonizacije bili su hospitalizacija na kliničkim odeljenjima, hospitalizacija preuzorkovanja duža od tri dana, primena cefalosporina i primena flourohinolona. U odnosu na odeljenja zahemodijalizu, boravak na odeljenjima za gerijatriju povećao je rizik za VRE kolonizaciju 6,5 puta,boravak u jedinicama intenzivnog lečenja 5 puta, a boravak na hemato-onkološkim odeljenjima 4,7 puta.U odnosu na ispitanike koji su hospitalizovani 48 sati pre uzorkovanja stolice na VRE, ispitanicihospitalizovani 3-7 dana pre uzorkovanja imali su 5,6 puta veći rizik za VRE kolonizaciju, ispitanicihospitalizovani 8-15 dana pre uzorkovanja 5,5 puta veći rizik za VRE kolonizaciju, dok su ispitanicihospitalizovani duže od 16 dana pre uzorkovanja imali 8,4 puta veći rizik za VRE kolonizaciju. Primenacefalosporina povećala je rizik za VRE kolonizaciju 2,2 puta, a primena flourohinolona 1,8 puta. Sviizolovani VRE sojevi su bili multirezistentni vanA Enterococcus faecium (VRfm) sojevi sa sledećimgenima za faktore virulencije (procenat sojeva s naznačenim genom je prikazan u zagradi): ermB-1(38,9%), esp (84%), efaA (71,2%), hyl (54,5%), asa1 (23,4%), gelE i cpd (11,6%) genima. Sposobnostproizvodnje biofilma pokazalo je 20,8% izolata. Analiza genetske srodnosti izolata otkrila je heterogenupopulaciju VRE izolata raspoređenih u 13 klastera sa 25 jedinstvenih MLVA profila (MT). VREfm sojevikoji su pripadali najvećim klasterima su bili dispergovani po različitim bolničkim odeljenjima. SRB3 jeoznačen kao osnivač populacije. SRB3 i SRB9 su označeni kao najbliži srodnici MT-1, a SRB16 kaonajbliži srodnik MT-159 klona...

INTRODUCTION: Intestinal carriage of vancomycin-resistant Enterococcus spp. (VRE) often precedes VRE infection and it could be of key importance in infection control. Our study represents the first epidemiological-microbiological study on VRE intestinal carriage in at-risk inpatients in high-risk departments for VRE colonization. AIMS: The aims of this study were: (1) to determine the frequency of VRE intestinal carriage among high-risk inpatients in Serbia in wards with an increased risk for VRE colonization; (2) identification of risk factors for VRE intestinal carriage; (3) examination of phenotypic and genotypic characteristics of isolated VRE strains and determination of clonal relatedness and clonal dissemination of circulating VRE strains. MATERIALS AND METHODS: The study population included 268 inpatients from six hospital departments at 3 university hospitals in Belgrade. To examine risk factors (RF) multivariate analysis was used. Characterization of the isolated VRE stains was performed with BD Phoenix system. Genotypic identification (ddlE. faecium, ddlE. faecalis), glycopeptide (vanA, vanB, vanC1, van C2/C3) and quinupristin– dalfopristin (Q–D) resistance probing (vatD, vatE, vgbA, ermB1), virulence gene detection (esp, hyl, efaA, asa1, gelE, cpd) and MLVA (Multiple-locus Variable-number tandem repeat analysis) were performed by molecular genetic methods. Biofilm formation was evaluated with the microtiter plate method. RESULTS: VRE carriage prevalence among at-risk patients was 28.7%. Independent predictors of VRE colonization among at-risk inpatients for VRE colonization were hospitalization in clinical wards, hospitalization longer than three days before sampling, use of cephalosporins and fluoroquinolones. Compared to the hemodialysis departments, stay in the geriatric departments increased the risk of VRE colonization 6.5 times, stay in intensive care units increased the risk 5 times, and a stay in the hematooncology departments 4.7 times. Compared to inpatients who were hospitalized 48 hours before stool sampling for VRE, inpatients hospitalized 3-7 days before sampling had 5.6-fold higher risk for VRE colonization, inpatients hospitalized 8–15 days prior to sampling had 5.5-fold higher risk for VRE colonization, while inpatients hospitalized longer than 16 days prior to sampling had 8.4-fold higher risk for VRE colonization. The use of cephalosporins increased the risk of VRE colonization 2.2 times and the use of fluoroquinolones 1.8 times. All VRE strains were vanA positive multidrug-resistant Enterococcus faecium (VRfm), harboring ermB-1 (38.9%), esp (84%), efaA (71.2%), hyl (54.5%), asa1 (23.4%), gelE and cpd (11.6%) genes. The ability of biofilm production was detected in 20.8%. Genetic relatedness of the isolates revealed 13 clusters and 25 unique MLVA (MT) profiles. VREfm strains belonging to the largest clusters were dispersed among different hospital departments. SRB3 was labeled as group founder. SRB3 and SRB9 were labeled as subgroup founders of MT-1 clone and SRB16 as subgroup founder of MT-159 clone. CONCLUSION: The obtained prevalence of VRE intestinal carriage among high-risk inpatients in Serbia is higher than European average. Prolonged hospitalization in high-risk departments and administration of cephalosporins and fluoroquinolones were the main risk factors for VRE colonization. High percentage of multidrug- resistance VREfm, with the ability of biofilm production and with various virulence genes that might affect the pathogenicity of the strains, as well as the emergence of resistance to Q–D which has never been licensed for clinical use in our country, are of particular concern. The illicit ABSTRACT usage of antibiotics in animal farming could be implicated. MLVA analysis revealed 29 genotypes, of which 25 were new. MLVA could exclude cross transmission among inpatients. Identified VREfm genotypes were phylogenetically closly related to the most common MTs causing VRE nosocomial infections in the world.