BE SS2024 Applied Microbiology 1 PDF

SS 2024 Lecture BE Applied Microbiology Prof. Dr. Joachim Fensterle Room 012-02-019 Phone -219 Mail [email protected] Applied Microbiology, Fensterle 1 General Information on Module Co...

SS 2024 Lecture BE Applied Microbiology Prof. Dr. Joachim Fensterle Room 012-02-019 Phone -219 Mail [email protected] Applied Microbiology, Fensterle 1 General Information on Module Contact time Self-study Lecture 30 h Preparation for contact 45 h time Lab course 30 h Literature review 20 h Preparation for exams 25 h Sum 60 h Sum 90 h  CP: 5  Lab course:  major part as a block course  No other lectures / lab courses in this time period. Applied Microbiology, Fensterle 2 1  SS 2024 Suggested Readings  Lecture slides:  moodle (PWD: BEAM-24)  YOUR LECTURE NOTES!  Suggested Literature:  Madigan, Martinko, Stahl, Clark: Brock Biology of Microorganisms Comprehensive microbiology textbook including metabolism pathways  Glazer, AN: Microbial Biotechnology: Fundamentals of Applied Microbiology Focus on industrial microbiological biotechnology  Antranikian, G: Angewandte Mikrobiologie German, targets most aspects of applied microbiology with focus on white biotechnology including metabolism pathways  Thieman, Palladino: Introduction to Biotechnology Suitable as first introduction for microbial biotechnology Applied Microbiology, Fensterle 3 Biotech-Bites erscheint am 17.4.2024 https://biotech-bites.com 4 2  SS 2024 Purpose of this lecture By this lecture, you should  know the importance of microorganisms in biotechnology.  know basic metabolic / fermentation pathways with relevance for applied microbiology.  expand your knowledge of distribution, characteristics and biotechnological and medical relevance of microorganisms.  recognize microorganisms as capable and efficient production systems for valuable chemical compounds and pharmaceuticals.  understand and apply basic biotechnological processes, in particular with respect to the metabolism of the selected microorganism. Applied Microbiology, Fensterle 5 You remember… If you see this sign… … you can feel like in cinema: lean back and enjoy  the content of this slide will not be relevant for the exam! However: it might still be helpful to look it up (in other words: your final goal is not just to pass the exam…) (Sign is copyrighted, source: 123RF). Applied Microbiology, Fensterle 6 3  SS 2024 General structure Applied Microbiology, Fensterle 7 Grading Applied Microbiology, Fensterle 8 4  SS 2024 Homework  4-5 original papers accompanying lecture  Self assessments on moodle  5 points if all self assessments are done, 3 points if 3/4 assessments are done.  Self assessment  grades of self assessment are just a feedback, points will be attributed if you have done the self assessment (regardless of the grade!). Applied Microbiology, Fensterle 9 Lab course Group selection etc:  see seperate Moodle course  REGISTER ASAP! Applied Microbiology, Fensterle 10 5  SS 2024 SS 2024 – Students from the Ukraine Lecture  For the students from the Ukraine, all lectures are available as recorded lectures  recorded lectures will be uploaded on youtube as private, the links will be provided to the students from the Ukraine Lab course  Blocked lab course weeks (2 weeks) in July, more information will follow SS 2023 Applied Microbiology, Fensterle 11 Overview Applied Microbiology, Fensterle 12 6  SS 2024 What is applied microbiology? Brock Biology of Microorganisms 13e © 2012 Pearson Applied Microbiology, Fensterle 13 Back to last semester…  We still have to talk about antimicrobial agents for in vivo use... Applied Microbiology, Fensterle 14 7  SS 2024 Chemical Growth Control Brock Biology of Microorganisms 13e © 2012 Pearson Applied Microbiology, Fensterle 15 Antimicrobial agents for in vivo use Synthetic antibacterial drugs  Growth factor analog  sulfa drugs (example: sulfanilamide) analog of p-aminobenzoic acid, precursor of folic acid  Isoniazid nicotinamide analog, specific for mycobacteria interferes with synthesis of mycolic acid (cell wall component) still most effective drug used against tuberculosis  Nucleic acid analogues  interfere with nucleic acid synthesis (usually pro- and eukaryotic!)  examples: 5-Fluorouracil  Quinolones  interfere with bacterial DNA gyrase, preventing supercoiling of DNA  still routinely used, e.g. drug of choice against anthrax infection Ciprofloxacin  example: ciprofloxacin Applied Microbiology, Fensterle 16 8  SS 2024 Antimicrobial agents for in vivo use Sulfa drugs Brock Biology of Microorganisms 13e © 2012 Pearson Applied Microbiology, Fensterle 17 Antimicrobial agents for in vivo use Antibiotics  Antibiotics are antimicrobial agents produced by microorganisms (generally fungi and bacteria)  < 1% of natural antibiotics useful in clinics  chemical modifications often used to enhance clinical efficacy = semi-synthetic antibiotics  important targets: ribosomes, cell wall, DNA replication, transcription, cytoplasmic membranes and lipid biosynthesis Applied Microbiology, Fensterle 18 9  SS 2024 Antimicrobial agents for in vivo use MoA of some antimicrobial agents Brock Biology of Microorganisms 13e © 2012 Pearson Applied Microbiology, Fensterle 19 Antimicrobial agents for in vivo use Annual production and use of antibiotics Brock Biology of Microorganisms 13e © 2012 Pearson Applied Microbiology, Fensterle 20 10  SS 2024 Antimicrobial agents for in vivo use Antibiotics – -lactams Penicillin G  Major representatives:  Penicillins (Penicillium sp.) N-acyl group  and Cephalosporins (Cephalosporium sp.)  both structures contain -lactam ring responsible for the MoA  inhibit cell wall synthesis by blocking transpeptidases, which catalyzes the cross-linking of two glycan linked peptide chains  resistance mechanism: cleavage of -lactam ring by -lactamases (bla genes)  natural -lactams mainly active against Gram positives  chemical modification broadens activity  activity against Gram negatives  resistance to -lactamases  Semi-synthetic cephalosporin cefriaxone antibiotic of choice for treatment of Neisseria gonorrhoeae infections, which are largely penicillin resistant Applied Microbiology, Fensterle 21 Antimicrobial agents for in vivo use Antibiotics – -lactams Natural and semi-synthetic Penicillins Brock Biology of Microorganisms 13e © 2012 Pearson Applied Microbiology, Fensterle 22 11  SS 2024 Antimicrobial agents for in vivo use Antibiotics - prokaryotic Aminoglycosides  contain amino-sugars linked by glycosidic linkage  target ribosomes inhibiting protein synthesis  examples: streptomycin (produced by Streptomyces sp.), kanamycin, neomycin, gentamicin  today, clinical use moderate, due to high toxicity and resistance Brock Biology of Microorganisms 13e © 2012 Pearson Applied Microbiology, Fensterle 23 Antimicrobial agents for in vivo use Erythromycin Antibiotics - prokaryotic macrolide ring Macrolides  clinically important broad spectrum antibiotics generally targeting sugars ribosomes  prominent example: erythromycin (Streptomyces sp.) Tetracyclines Tetracyclin  clinically important broad spectrum antibiotics produced by Streptomyces sp.  target ribosomes  inactivated by range of cations, including Ca2+  no intake with milk Wikipedia, Brock Biology of Microorganisms 13e © 2012 Pearson Applied Microbiology, Fensterle 24 12  SS 2024 Antimicrobial drug resistance Cartoon by Nick Kim, www.nearingzero.net Applied Microbiology, Fensterle 26 Antimicrobial drug resistance Bacterial resistance Mechanism Antibiotic Genetic basis Present in (expl.): example Reduced permeability Penicillin G chromosomal Gram negatives Inactivation of Penicillins plasmid and Staph. aureus antibiotic Chloramphenicol chromosomal Neisseria (e.g. -lactamase, gonorrhoeae methylases, acetylases, Staph. aureus phosphorylases) Aminoglycosides Staph. aureus Alteration of target Erythromycin chromosomal Staph. aureus (e.g. RNA polymerase, Rifamycin Enteric Bacteria ribosome, DNA gyrase) Streptomycin Enteric Bacteria Norfloxacin Enteric Bacteria Staph. aureus Develoment of resistant Sulfonamides chromosomal Enteric Bacteria biochemical pathway Staph. aureus Efflux Tetracyclines plasmid Enteric Bacteria (pumping out of cells) Chloramphenicol chromosomal Bacillus subtilis Staph. aureus Applied Microbiology, Fensterle 27 Erythromycin chromosomal Staph. aureus 13  SS 2024 Antimicrobial drug resistance Bacterial resistance Inactivation of antibiotics mediated by enzymes encoded by R plasmid genes Brock Biology of Microorganisms 13e © 2012 Pearson Applied Microbiology, Fensterle 28 Antibiotics – Antimicrobial agents for in vivo use Appearance of antimicrobial resistance in pathogens Relationship between use and resistance !! !! !! !! Applied Microbiology, Fensterle!!: strains with resistance to 29 all known antimicrobial drugs isolated Brock Biology of Microorganisms 13e © 2012 Pearson 14  SS 2024 Assessing antimicrobial activities Minimal inhibitory concentration MIC:  Smallest amount of agent needed to inhibit growth of test organism  Assessed by tube dilution technique:  incubation of given organism with serial diluted agent  Tubes are checked for visual growth (turbidity)  MIC = lowest concentration with complete block  To be comparable, assay must be standardized (inoculum, temperature, pH, areation…) Applied Microbiology, Fensterle 30 Assessing antimicrobial activities Diffusion methods:  discs with antimicrobial agent placed on plates inoculated with test organisms  agent diffuses to agar, diameter of inhibition proportional to MIC  routinely used to test sensitivity of bacteria against antibiotics Brock Biology of Microorganisms 13e © 2012 Pearson Applied Microbiology, Fensterle 31 15  SS 2024 Example from the lab: gentamicin German-French GC „Signal transduction, where cancer and infection converge“ Caspase-1 as a target of bacterial tumour therapy Katharina Galmbacher PhD thesis, supervisor: J. Fensterle Applied Microbiology, Fensterle 32 Purification of macrophages tumor tumor cell other cells (fibroblasts, endothelial cells,…) tumor associated macrophages bacteria Applied Microbiology, Fensterle 33 16  SS 2024 Purification of macrophages Total cell suspension Total cell suspension MACS® +Genta - Genta + - + Genta Macrophages Macrophages depleted Cfu & Western blot for caspase-1 expression and Applied Microbiology, Fensterle 34 activation Purification of macrophages Macrophages Macrophages depleted Applied Microbiology, Fensterle 35 17  SS 2024 Result: Salmonella loc. after iv infection of tumor bearing mice Salmonella in tumors spleen-Genta spleen+Genta Total cells-Genta Total cells+Genta Macrophages+Genta Macrophages depleted+Genta Applied Microbiology, Fensterle 36 Lesson: don‘t trust what others say! Number of intracellular Number of bacteria after bacteria 1 h after incubation Genta-incubation with Genta  MIC values tells how much antibiotics you need to block growth (not kill!) in 12 hours  100 µg usually used in literature are not enough  300 µg/ml Genta are sufficient to reduce extracellular bacteria >1000x within one hour and do not show substantial killing of intracellular bacteria Applied Microbiology, Fensterle 37 18  SS 2024 Perfect! The detailed protocol:  Incubate whole cell suspension with 300 µg/ml gentamycin for 1 hour at 37°C  In parallel, perform MACS cell separation in the presence of 300 µg genta on ice (due to staining procedure)  Afterwards, determine CFU Applied Microbiology, Fensterle 38 Lesson 2: Don‘t trust what you say! CFU after 1 h incubation with 300 µg/ml  Gentamycin is genta at different temperatures completely inactive in killing bacteria at 4°C  We had to repeat the in vivo experiment with Salmonella and perform the cell separation after 1h preincubation at 37°C  Applied Microbiology, Fensterle 39 19