The effect of heavy metal ions on Staphylococcus aureus, complex biochemical and MALDI-TOF study. Dagmar Chudobová
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1 The effect of heavy metal ions on Staphylococcus aureus, complex biochemical and MALDI-TOF study Dagmar Chudobová
2 2 Staphylococcus aureus G + bacteria of the genus Staphylococcus (Ø 1 μm) often cause of infections pathogenic - production of toxins in 1/3 of the population naturally presented on the skin and mucous membranes may be presented in salami, pork, potato salad and ice cream
3 3 Heavy metals Metals density higher than 5 g.cm -3 Used heavy metals for the experiment Ag - neurotoxin, cumulation in the heart Cd a proven carcinogen, cumulation in the lungs, liver and bones Cu essential for human, abundance cumulation in the liver
4 4 Heavy metals Pb accumulative toxin, 95 % of it remains in the bones, in children particularly in the brain - one of the causes of osteoporosis and tooth decay - overload the kidney, cause fatigue, impairs memory, cause hormonal disorders, supports the creation of multiple sclerosis Zn important for the IS and as part of antioxidant processes - chronic poisoning isn t described - anemia, damage of the pancreas
5 The effect of heavy metal ions on cells 5
6 6 Aims of study Monitoring of the effect of heavy metal ions on the properties of S.aureus strains resistant to the effect of heavy metal ions (Ag, Cd, Cu, Pb, Zn) Comparison to control non-resistant strain of S.aureus Used methods: microscopy, microbiology, biochemistry, mass spectrometry, electrochemistry, molecular biology
7 Results - microscopy (highest concentrations) 7 S.a. Ag Cd Cu Pb Zn
8 Viability (%) Viability (%) Viability (%) Viability (%) Viability (%) Absorbance (AU) Absorbance (AU) Absorbance (AU) Absorbance (AU) Absorbance (AU) Results microbiology (highest 8 concentrations) A 1,000 0,800 Ag 1,000 0,800 Cu 1,000 0,800 Cd 1,000 0,800 Zn 1,000 0,800 Pb C Heavy metal ions Time of measurement IC 50 (μm) B 0,600 0,400 0,200 0, % 100% 80% 60% 40% 20% 0% Time (h) Concentration of Ag (µm) 0,600 0,400 0,200 0, % 100% 80% 60% 40% 20% 0% Time (h) Concentration of Cu (µm) 0,600 0,400 0,200 0, % Time (h) 0,600 0,400 0,200 0, Time (h) a b c d e 100% 80% 60% 40% 20% 0% Concentration of Cd (µm) 120% 100% 80% 60% 40% 20% 0% Concentration of Zn (µm) 0,600 0,400 0,200 0, % 100% 80% 60% 40% 20% 0% Time (h) Concentration of Pb (µm) Ag 6 h h h h 72.1 Cu 6 h h h h 1.0 Cd 6 h h h h 14.1 Zn 6 h h h h 88.6 Pb 6 h h h h 1.0
9 Change in enzymatic activity (μkat/l) GAE (μg/ml) GAE (μg/ml) Change in enzymatic activity (μkat/l) Change in enzymatic activity (μkat/l) Change in enzymatic activity (μkat/l) Results spectrophotometry (highest concentrations) 9 ALT (µkat/l) AST (µkat/l) ALP (µkat/l) 0,1 0,08 0,06 0,04 0,02 0-0,02-0,04 Ag Cu Pb Zn Cd Ag Cu Pb Zn Cd 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0-0,2 Ag Cu Pb Zn Cd GMT (µkat/l) FRAP ABTS 0,6 0,5 0,4 0,3 0,2 0,1 0-0,1-0,2 Ag Cu Pb Zn Cd 0,6 0,5 0,4 0,3 0,2 0,1 0 Ag Cu Pb Zn Cd Ag Cu Pb Zn Cd
10 Concentration of GSH (μm) Concentration of GSSG (μm) Ammount of MT (μm) A na Results electrochemistry, HPLC S.a. Ag Cu Pb Zn Cd (highest concentrations) g B a scan c scan e 1000 na na na Potential (V) Potential (V) b d na scan f 1000 na scan Potential (V) 1000 na Potential (V) C Potential (V) a GSH scan S.a. Ag Cu Pb Zn Cd Potential (V) b scan Potential (V) GSSG S.a. Ag Cu Pb Zn Cd
11 S.a. Ag Results - MALDI-TOF (highest concentrations) aureus DSM 4910 Staphylococcus simulans DSM Cd Cu aureus DSM Staphylococcus saprophyticus ssp saprophyticus CCM 2682 Pb Staphylococcus simulans DSM Zn Staphylococcus felis DSM 7377T
12 Results RFLP (highest concentrations) 12 Sequencing S.a. Staphylococcus aureus Ag Bacillus punilus/safarensis Cd Staphylococcus aureus Cu Bacillus licheniformis Pb Bacillus punilus/altitudinis Zn Bacillus licheniformis
13 Results RFLP, MALDI-TOF, microscopy (100 µm) 13 S.a. aureus DSM DSM S.a. Sequencing Ag Staphylococcus aureus Cd Staphylococcus aureus Cu Staphylococcus aureus Pb Staphylococcus aureus Zn Staphylococcus aureus Ag Cd Cu Pb aureus DSM DSM aureus DSM 4910 DSM aureus DSM 4910 DSM aureus DSM 4910 DSM Ag Cd Cu Pb Zn aureus DSM 4910 DSM Zn
14 Results RFLP, MALDI-TOF, microscopy (150 µm) 14 S.a. aureus DSM DSM S.a. Sequencing S.a. Staphylococcus aureus Ag Bacillus punilus/safarensis Cd Staphylococcus aureus Cu Bacillus licheniformis Pb Bacillus punilus/altitudinis Zn Bacillus licheniformis Ag Cd Cu Pb aureus DSM DSM aureus DSM 4910 DSM Staphylococcus auricularis DSM DSM Staphylococcus simulans DSM Ag Cd Cu Pb Zn Staphylococcus hominis ssp novobiosepticus DSM 15614T DSM Zn
15 15 Conclusion Significant changes in the morphological, biochemical, genetic and proteomic properties Description of the basic analysis of resistant strains of S.aureus Basic for further studies of resistant strains Publication of the results in the Journal of Microbiology, International Journal of Enviromental Research and Public Health or??? 15
16 Acknowledgment Bc. Simona Dostálová Msc. Miguel Angel Merlos Rodrigo Mgr. Darina Mašková Ing. Kateřina Tmejová, Ph.D. Ing. Iva Blažková Prof. Ing. René Kizek, Ph.D. Support from PGS11_2012 The effect of heavy metal ions on the biological properties, resistance and virulence 16
17 Thank you for your attention 17
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