Генотипизација, резистенција на антибиотике и формирање биофилма клиничких изолата Stenotrophomonas maltophilia из Србије

Stenotrophomonas maltophilia is an environmental bacterium and an opportunistic pathogen usually associated with healthcare-associated infections, which has recently been recognized as a global multidrug resistant organism. The aim of this study was genotyping and physiological characterization of S. maltophilia collected during the routine health care at The Institute for Mother and Child Health Care of Serbia “Dr Vukan Čupić“. It is the large, tertiary care pediatric hospital in Belgrade, Serbia, hosting the national reference cystic fibrosis (CF) center for pediatric and adult patients. We characterized 88 S. maltophilia strains, 42 strains of cystic fibrosis (CF) and 46 strains of non-cystic fibrosis (non-CF) origin isolated from 2013 to 2015 in order to investigate their genetic relatedness and phenotypic traits. Genotyping was performed using sequencing of 16S rRNA gene, Pulse Field Gel Electrophoresis (PFGE) and Multi locus sequencing typing (MLST) analysis. Sensitivity to five relevant antimicrobial agents was determined, namely trimethoprim/sulfamethoxazole (TMP/SMX), chloramphenicol, ciprofloxacin, levofloxacin and tetracycline. Surface characteristics, motility, biofilm formation and adhesion to mucin were tested in all strains. In addition, influence of different factors (temperature, pH, agitation and CO2) on biofilm formation, kinetics of selected biofilm producers and effect of TMP/SMX on formed biofilm were analyzed. Statistical approach was used to determine correlations between obtained results. All analyzed clinical isolates belong to S. maltophilia species with identity ranging from 95% to 99% with S. maltophilia strains from the NCBI database what was confirmed by sequencing of PCR products of amplified 16S rRNA gene..

Temperature, pH and Trimethoprim-Sulfamethoxazole Are Potent Inhibitors of Biofilm Formation by Stenotrophomonas maltophilia Clinical Isolates

Stenotrophomonas maltophilia, an opportunistic pathogen usually connected with healthcare-associated infections, is an environmental bacterium. Intrinsic resistance to multiple antibiotics, with different virulence determinants in the last decade classified this bacterium in the group of global multiple drug resistant (MDR) organism. S. maltophilia clinical isolates, were collected from tertiary care pediatric hospital in Belgrade, Serbia to investigate influence of different factors on biofilm formation, kinetics of biofilm formation for strong biofilm producers and effect of trimethoprim-sulfamethoxazole (TMP/SMX) on formed biofilm. Most of the isolates (89.8%) were able to form a biofilm. Analysis of biofilm formation in different growth conditions showed that changing of temeperature and pH had the stronggest effect on biofilm formation almost equally in group of cystic fibrosis (CF) and non-CF strains. TMP/SMX in concentration of 50 mu g/ml reduced completely 24 h old biofilms while concentration of 25 mu g/ml effects formed biofilms in a strain dependent manner. Among strains able to form strong biofilm CF isolates formed biofilm slower than non-CF isolates, while shaking conditions did not affect biofilm formation. Swimming motility was detected in both CF and non-CF isolates, however more motile strain formed stronger biofilms. This study suggests that temperature, pH and TMP/SMX had the strongest influence on biofilm formation in analyzed collection of S. maltophilia. A positive correlation between motility and strength of formed biofilm was demonstrated

Genotypic and Phenotypic Characterization of Stenotrophomonas maltophilia Strains from a Pediatric Tertiary Care Hospital in Serbia

Background Stenotrophomonas maltophilia is an environmental bacterium and an opportunistic pathogen usually associated with healthcare-associated infections, which has recently been recognized as a globally multi-drug resistant organism. The aim of this study was genotyping and physiological characterization of Stenotrophomonas maltophilia isolated in a large, tertiary care pediatric hospital in Belgrade, Serbia, hosting the national reference cystic fibrosis (CF) center for pediatric and adult patients. Methods We characterized 42 strains of cystic fibrosis (CF) and 46 strains of non-cystic fibrosis (non-CF) origin isolated from 2013 to 2015 in order to investigate their genetic relatedness and phenotypic traits. Genotyping was performed using sequencing of 16S rRNA gene, Pulse Field Gel Electrophoresis (PFGE) and Multi locus sequencing typing (MLST) analysis. Sensitivity to five relevant antimicrobial agents was determined, namely trimethoprim/sulfamethoxazole (TMP/SMX), chloramphenicol, ciprofloxacin, levofloxacin and tetracycline. Surface characteristics, motility, biofilm formation and adhesion to mucin were tested in all strains. Statistical approach was used to determine correlations between obtained results. Results Most of the isolates were not genetically related. Six new sequence types were determined. Strains were uniformly sensitive to all tested antimicrobial agents. The majority of isolates (89.8%) were able to form biofilm with almost equal representation in both CF and non-CF strains. Swimming motility was observed in all strains, while none of them exhibited swarming motility. Among strains able to adhere to mucin, no differences between CF and non-CF isolates were observed. Conclusions High genetic diversity among isolates implies the absence of clonal spread within the hospital. Positive correlation between motility, biofilm formation and adhesion to mucin was demonstrated. Biofilm formation and motility were more pronounced among non-CF than CF isolates

Temperature, pH and Trimethoprim-Sulfamethoxazole Are Potent Inhibitors of Biofilm Formation by Stenotrophomonas maltophilia Clinical Isolates

Stenotrophomonas maltophilia, an opportunistic pathogen usually connected with healthcare-associated infections, is an environmental bacterium. Intrinsic resistance to multiple antibiotics, with different virulence determinants in the last decade classified this bacterium in the group of global multiple drug resistant (MDR) organism. S. maltophilia clinical isolates, were collected from tertiary care pediatric hospital in Belgrade, Serbia to investigate influence of different factors on biofilm formation, kinetics of biofilm formation for strong biofilm producers and effect of trimethoprim-sulfamethoxazole (TMP/SMX) on formed biofilm. Most of the isolates (89.8%) were able to form a biofilm. Analysis of biofilm formation in different growth conditions showed that changing of temeperature and pH had the stronggest effect on biofilm formation almost equally in group of cystic fibrosis (CF) and non-CF strains. TMP/SMX in concentration of 50 μg/ml reduced completely 24 h old biofilms while concentration of 25 μg/ml effects formed biofilms in a strain dependent manner. Among strains able to form strong biofilm CF isolates formed biofilm slower than non-CF isolates, while shaking conditions did not affect biofilm forma­tion. Swimming motility was detected in both CF and non-CF isolates, however more motile strain formed stronger biofilms. This study suggests that temperature, pH and TMP/SMX had the strongest influence on biofilm formation in analyzed collection of S. maltophilia. A positive correlation between motility and strength of formed biofilm was demonstrated

Adhesion of <i>S</i>. <i>maltophilia</i> isolates to mucin-coated and non-coated wells of microtiter plate.

<p>Bars represent mean values ± standard errors.</p

Biofilm formed by individual bacterial <i>Stenotrophomonas maltophilia</i> strains isolated from CF and non-CF patients.

<p>Biofilm formed by individual bacterial <i>Stenotrophomonas maltophilia</i> strains isolated from CF and non-CF patients.</p

Antibiotic susceptibility of the <i>S</i>. <i>maltophilia</i> clinical isolates (n = 88).

<p>Antibiotic susceptibility of the <i>S</i>. <i>maltophilia</i> clinical isolates (n = 88).</p

Genotypic and Phenotypic Characterization of <i>Stenotrophomonas maltophilia</i> Strains from a Pediatric Tertiary Care Hospital in Serbia

<div><p>Background</p><p><i>Stenotrophomonas maltophilia</i> is an environmental bacterium and an opportunistic pathogen usually associated with healthcare-associated infections, which has recently been recognized as a globally multi-drug resistant organism. The aim of this study was genotyping and physiological characterization of <i>Stenotrophomonas maltophilia</i> isolated in a large, tertiary care pediatric hospital in Belgrade, Serbia, hosting the national reference cystic fibrosis (CF) center for pediatric and adult patients.</p><p>Methods</p><p>We characterized 42 strains of cystic fibrosis (CF) and 46 strains of non-cystic fibrosis (non-CF) origin isolated from 2013 to 2015 in order to investigate their genetic relatedness and phenotypic traits. Genotyping was performed using sequencing of 16S rRNA gene, Pulse Field Gel Electrophoresis (PFGE) and Multi locus sequencing typing (MLST) analysis. Sensitivity to five relevant antimicrobial agents was determined, namely trimethoprim/sulfamethoxazole (TMP/SMX), chloramphenicol, ciprofloxacin, levofloxacin and tetracycline. Surface characteristics, motility, biofilm formation and adhesion to mucin were tested in all strains. Statistical approach was used to determine correlations between obtained results.</p><p>Results</p><p>Most of the isolates were not genetically related. Six new sequence types were determined. Strains were uniformly sensitive to all tested antimicrobial agents. The majority of isolates (89.8%) were able to form biofilm with almost equal representation in both CF and non-CF strains. Swimming motility was observed in all strains, while none of them exhibited swarming motility. Among strains able to adhere to mucin, no differences between CF and non-CF isolates were observed.</p><p>Conclusions</p><p>High genetic diversity among isolates implies the absence of clonal spread within the hospital. Positive correlation between motility, biofilm formation and adhesion to mucin was demonstrated. Biofilm formation and motility were more pronounced among non-CF than CF isolates.</p></div

Phylogenetic analysis of obtained PFGE <i>Xba</i>I profiles of <i>S</i>. <i>maltophilia</i> clinical isolates.

<p>Distance showed above the dendrogram represents genetic relatedness between the analyzed strains. MLST analysis represents strains used for this analysis and obtained results. Patient number represents strains obtained from CF and non-CF patients, C and N, respectively. Isolates obtained from the same patients have identical patients number. Source represents site of isolation: R–respiratory tract, B–blood, HM–human milk. Strength of biofilm formed is presented with—–no biofilm, +–weak biofilm, ++–medium biofilm, +++–strong biofilm.</p