Inhibition of Escherichia coli growth by adherence to saponite particles

Transcription

1 HDID V How dead is dead? University Clinic of Dentistry, September 6-8, 2017 Vienna, Austria Inhibition of Escherichia coli growth by adherence to saponite particles 1 Gaálová Barbora, 1 Vyletelová Ivana, 2 Judith Kikhney 2 Annette Moter, 1 Helena Bujdáková 1 Comenius University in Bratislava, Faculty of Natural Sciences, Department of Microbiology and Virology, Ilkovičova , Bratislava, Slovakia 2 University Medical Center Berlin, Biofilmcenter at the German Heart Institute Berlin, Germany

2 Clay minerals Structure and effect on microorganisms natural nanomaterials (< 2 µm) high absorption capacity large surface area ion exchange capacity general use - mainly as adsorbents of chemical substances - carrier for bioactive substances

3 Goals of research Is saponite effective against bacteria? Which is the mechanisms of action? Saponit (Sumecton, Kunimine Ind., Japan) smectite clay multilayered silicate structure negatively charged c1 (5 mg/ml); c2 (2.5 mg/ml) and c3 (1.25 mg/ml) model system gramnegative E. coli

4 Identification of E. coli isolates E. coli CCM 3954 (Brno, Czech Republic) clinical isolates from uro-infections environmental isolates - identification by biochemical tests - molecular confirmation Characteristic growth of E. coli strains on diagnostic media: Endo agar (a), MacConkey agar (b), Chrom agar (c); Example of species specific restriction of PCR products of 16S rdna (a) using enzymes BstBI (b) and HaeIII (c; according to Lu et al. 2000).

5 Characterization of E. coli isolates antimicrobial susceptibility testing (disc diffusion method, EUCAST 2017) ability to form biofilm (Chang et al. 2010; 0.1 % crystal violet staining) Determination of antibiotic susceptibility of uro-isolates of E. coli (fig. 1) and enviro-isolates (fig. 2); Determination of biofilm formation of uro-isolates and enviro-isolates (fig. 3)

6 Antimicrobial effect of saponite to E. coli CCM 3954 viability assay using MTT reduction plating samples on Mueller-Hinton agar, CFU visualization of attachment and viability of cells CLSM with propidium iodide, DAPI and Resazurine (Alamar Blue) staining Figure shows results obtained by MTT (fig. 4) and CFU ( fig. 5) method after incubation of the E. coli CCM 3954 in the presence of different concentrations of saponite (upper graphs). Figure from light microscopy (a) shows the reference strain E. coli CCM 3954 after staining of death cells with propidium iodide. Figure from CLSM (b) shows the reference strain E. coli CCM 3954 after staining of viable cells with Resasurine (Alamar Blue). Both pictures were performed in the presence of 5 mg/ml of saponite and incubation was 24 h at 37 C.

7 Antimicrobial effect of saponite to E. coli CCM 3954 MTT assay, CFU and FISH with 16S rrna labelling probe - detection of attachment, abundance and metabolic activities of adhered cells

8 Effect of saponite to E. coli CCM 3954 MTT assay, CFU and FISH with 16S rrna labelling probe

9 Effect of saponite to E. coli CCM 3954 SEM analysis

10 Effect of saponite to E. coli isolates uro-isolate 16 and enviro-isolate KP2 Detection of 4 genes encoding adhesines fima, pap, afa, aaf Example of FimA detection: 1. E.coli CCM 3954; 2. uro-isolate 9; 3. uro-isolate 26; 4. enviroisolate KP2, KP3, KP4, NTC, 1 kbp ladder

11 Conclusions The concentration of 5 mg/ml of saponite inhibited growth of E. coli different response of uro-isolates and enviro-isolates to saponite adhesines FimA, pap ability to form biofilms other tested gramnegative and grampositive bacteria antimicrobial effect possible mechanisms - physical capturing mechanism involving electrochemical interactions of cell wall/plasmatic membrane and clay suface - E. coli adheres (through adhesines) to saponite particles, thus its growth is inhibited and cells are unable to divide - cells embedded in deeper layers of saponite, demage of cell wall

12 Acknowledgements ipromedai, COST action TD1305 STSM-TD Visualization of adhered Escherichia coli strains to clay particles using Fluorescent in situ hybridization Slovak Research and Development Agency under the contract No. [APVV ] Prevention and eradication of microbial biofilms in relation to nanomaterials Thank you for attention