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

Genotyping of Bacillus spp. isolate collection from natural samples

The aim of this study was genotyping and identification of collection of 164 Bacillus spp. isolates, from samples of soil, manure, and straw gathered from across Serbia, using Pulse field gel electrophoresis (PFGE) combined with sequencing of tuf gene, one of the housekeeping genes. The PFGE analysis with NotI enzyme was used to determine phylogenetic relationships of isolates and referent strains. Four large groups of Bacillus spp. were distinguishable: cereus, subtilis, pumilus and megaterium and within enormous genetic diversity. Bacillus subtilis Marburg referent strain did not group with rest of the strains from the subtilis group (Bacillus subtilis ATCC6633 and Bacillus atrophaeus ATCC9372). Strains from the cereus group were distinguished and closely grouped together. One representative isolate from each of 21 distinct PFGE groups was identified by sequencing of tuf gene. Eight different species were identified among chosen isolates: B. amyloliquefaciens, B. subtilis, B. pumilus, B. safensis, B. megaterium, B. cereus, B. anthracis and B. thuringiensis. Our results showed that PFGE analysis combined with sequencing of one of the housekeeping genes could be used for characterization of large collections of Bacillus isolates. The determination of tuf gene recommended itself to be an adequate and sufficient analysis for obtaining very clear and unambiguous results, with high resolution of separation of Bacillus species. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173026

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

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

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

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

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