Fenotipska i genotipska karakterizacija bakterija Burkholderia cepacia kompleksa izolovanih kod pacijenata sa cističnom fibrozom

Introduction: The respiratory tract impairment due to chronic microbial colonization and infection is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. It is estimated that over 80% of individuals with CF die directly or indirectly from pulmonary disease. The Burkholderia cepacia complex (Bcc) is one of the most important groups of organisms causing infection in the CF lung, associated with a dubious prognosis and patientto-patient transmissibility. Respiratory tract infection may persist for months or years or be associated with acute life-threatening illness. Since the first cases were described in the late 1970s and early 1980s, the reported prevalence of Bcc in CF population varied between different countries and CF care centres reaching up to 30%, e.g. in the Czech Republic and the United Kingdom due to the epidemic spread. The highly transmissible and virulent clonal lineages associated with outbreaks belong to B. cenocepacia, and the most successful are ET12 (ST28), CZ1 (ST32), PHDC and Midwest clones. Although most species within Bcc are potentially capable of causing infections in CF patients, B. cenocepacia and B. multivorans are significantly more common than the remaining species and together they account for around 70% of all Bcc infections in the course of CF. Since the early 2000s the epidemiology of the Bcc has changed. In many Western European countries as well as in Canada and the United States, overall Bcc prevalence rates decreased and now range from 1.4% to around 6% as a result of effective prevention strategies. Notwithstanding, data regarding prevalence and epidemiology of Bcc in countries on the Balkan Peninsula is scarce. Objectives: The aim of this study was to evaluate the prevalence, epidemiological characteristics, presence of molecular markers associated with virulence and transmissibility, antimicrobial susceptibility and biofilm formation capacity of the Bcc strains in the National Cystic Fibrosis Centre which is located at the Mother and Child Health Care Institute of Serbia “Dr. Vukan Cupic”, a university-affiliated tertiary care paediatric hospital in Belgrade. Materials and methods: The Bcc isolates collected during the four-year study period (2010– 2013) were further examined by 16s rRNA gene, recA PCR, pulsed-field gel electrophoresis of genomic DNA, multilocus sequence typing analysis, and phylogenetic analysis based on concatenated sequence of seven alleles. Selected isolates, previously characterized with respect to the species status and the genotype, were tested for their susceptibility to seven antimicrobials using the E-test method. PCR screenings of eight genetic determinants (cblA, BCESM, cepI, cepR, fliG, llpE, wbiI, bcscV) associated with virulence were performed on genomic DNA extracted from the 14 isolates representative of all genotypes. Results: Fifty out of 184 patients (27.2%) were colonized with two Bcc species, B. cenocepacia (n=49) and B. stabilis (n=1). Thirty-four patients (18.5%) had chronic colonization. Typing methods revealed a high level of similarity among B. cenocepacia isolates, indicating a person-to-person transmission or acquisition from a common source. New sequence types (STs) were identified, and none of the STs with an international distribution were found. One centre-specific ST, B. cenocepacia ST856, was highly dominant and shared by 48/50 (96%) patients colonized by Bcc. This clone was characterized by PCR positivity for both the B. cepacia epidemic strain marker and cable pilin, and showed close genetic relatedness to the epidemic strain CZ1 (ST32). All patients harboured genetically similar strains throughout the period of observation. Meropenem and ceftazidime were the most effective antimicrobials tested in vitro, with 72.6% and 71.6% of the isolates being susceptible to these compounds, respectively. A significant variation of the antimicrobial susceptibility pattern of clonal isolates obtained from persistently infected patients was registered. The majority of the Bcc isolates (88%) were capable of forming biofilm in vitro. Conclusions: These results indicate that the impact of Bcc on airway colonization in the Serbian CF population is high and virtually exclusively limited to a single clone of B. cenocepacia. The presence of a highly transmissible clone and probable patient-to-patient spread was observed.