Microbiology. Zhenmei Lu ( 吕镇梅 ) 2017 Spring-Summer

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1 Microbiology Zhenmei Lu ( 吕镇梅 ) 2017 Spring-Summer

2 Chapter 26 Microbial Growth Control

3 Outline I. Physical Antimicrobial Control II. Chemical Antimicrobial Control III. Antimicrobial Agents Used In Vivo IV. Control of Viruses and Eukaryotic Pathogens V. Antimicrobial Drug Resistance and Drug Discovery

4 I. Physical Antimicrobial Control 26.1 Heat Sterilization 26.2 Radiation Sterilization 26.3 Filter Sterilization

5 26.1 Heat Sterilization Sterilization The killing or removal of all viable organisms within a growth medium Inhibition Effectively limiting microbial growth Decontamination The treatment of an object to make it safe to handle Disinfection Directly targets the removal of all pathogens, not necessarily all microorganisms

6 Survival fraction (log scale) 26.1 Heat Sterilization The effect of temperature over time on the viability of a mesophilic bacterium C Decimal reduction time (D) 50 C 60 C Time (min)

7 Decimal reduction time (min) 26.1 Heat Sterilization Relationship between temperature and the rate of killing in mesophiles and thermophiles (a) (b) Temperature ( C)

8 26.1 Heat Sterilization Chamber pressure gauge Steam exhaust valve Steam exhaust Door Thermometer and valve Jacket chamber Air exits through vent Steam supply valve Steam enters here The Autoclave

9 26.1 Heat Sterilization The Autoclave

10 Temperature ( C) 26.1 Heat Sterilization Autoclave time 130 Stop steam Begin pressure Sterilization time 100 Flowing steam Temperature of object being sterilized Temperature of autoclave Total cycle time (min) The moist heat sterilization Pasteurization

11 26.2 Radiation Sterilization Microwaves, UV, X-rays, gamma rays, and electrons can reduce microbial growth --UV A biological safety cabinet

12 26.2 Radiation Sterilization --Ionizing Radiation Electrons, hydroxyl radicals, and hydride radicals

13 Survival fraction (log scale) 26.2 Radiation Sterilization D10 10% survival 0.01 Radiation (Grays) Relationship between the survival fraction and the radiation dose

14 26.2 Radiation Sterilization

15 26.3 Filter Sterilization A depth filter (HEPA) A Membrane filter A Nucleopore filter

16 26.3 Filter Sterilization

17 26.3 Filter Sterilization

18 II. Chemical Antimicrobial Control 26.4 Chemical Growth Control 26.5 Chemical Antimicrobial Agents for External Use

19 Log cell number Log cell number Log cell number 26.4 Chemical Growth control Bacteriostatic Total cell count Bacteriocidal Total cell count Viable cell count Viable cell count Time Time Bacteriolytic Total cell count Viable cell count Time

20 26.4 Chemical Growth control -- MIC by tube dilution technique

21 26.4 Chemical Growth control -- Measuring antimicrobial activity by disk diffusion technique Nutrient agar plate Antibiotic discs are placed on surface Inoculate plate with a liquid culture of a test organism Incubate for h Test organism shows susceptibility to some antibiotics, indicated by inhibition of bacterial growth around discs (zones of inhibition) after incubation

22 丁胺卡那霉素 头胞菌素 氯霉素 硫酸庆大霉素 氨苄青霉素 克林霉素 先锋霉素 青霉素钠 红霉素

23 26.5 Chemical Antimicrobial Agents -- External Use

24 26.5 Chemical Antimicrobial Agents -- External Use

25

26 III. Antimicrobial Agents Used In Vivo 26.6 Synthetic Antimicrobial Drugs 26.7 Naturally Occurring Antimicrobial Drugs: Antibiotics Lactam Antibiotics: Penicillins and Cephalosporins 26.9 Antibiotics from Prokaryotes

27 Chemical Antimicrobial Agents -- In Vivo Use Animation: Antibiotic Modes of Action

28 Chemical Antimicrobial Agents -- In Vivo Use Eukaryotes Bacteria Obligately parasitic Bacteria Viruses Fungi Mycobacteria Gram-negative Bacteria Gram-positive Bacteria Chlamydia Rickettsia RNA viruses DNA viruses Azoles Allylamines Cycloheximide Polyenes Polyoxins Nucleic acid analogs Echinocandins Isoniazid Tobramycin Streptomycin Polymyxins Penicillins Sulfonamides Cephalosporins Quinolones Vancomycin Daptomycin Platensimycin Tetracycline Nonnucleoside reverse-transcriptase inhibitors Protease inhibitors Fusion inhibitors Nucleoside analogs Interferon Antimicrobial spectrum of activity

29 Chemical Antimicrobial Agents -- In Vivo Use Annual worldwide production and use

30 Chemical Antimicrobial Agents -- Synthetic Antimicrobial Drugs Salvarsan one of the first useful antimicrobial agents Paul Ehrlich, 1900s

31 Chemical Antimicrobial Agents -- Synthetic Antimicrobial Drugs Sulfa drugs Sulfanilamide p-aminobenzoic acid Folic acid Gerhard Domagk, 1930s

32 Growth factor Analog Synthetic Antimicrobial Drugs Phenylalanine (an amino acid) p-fluorophenylalanine Uracil (an RNA base) 5-Fluorouracil (a uracil analog) Ciprofloxacin, a quinolone antimicrobial drug Thymine (a DNA base) 5-Bromouracil (a thymine analog)

33 Antibiotics -Lactam Designation NATURAL PENICILLIN Benzylpenicillin (penicillin G) Gram-positive activity -lactamase-sensitive N-Acyl group SEMISYNTHETIC PENICILLINS N-Acyl group -Lactam ring Thiazolidine ring 6-Aminopenicillanic acid Methicillin acid-stable, -lactamase-resistant Oxacillin acid-stable, -lactamase-resistant Dihydrothiazine ring Ampicillin broadened spectrum of activity (especially against gram-negative Bacteria), acid-stable, -lactamase-sensitive -lactam ring Ceftriaxone Carbenicillin broadened spectrum of activity (especially against Pseudomonas aeruginosa), acid-stable but ineffective orally, -lactamase-sensitive

34 Antibiotics from Prokaryotes -- Aminoglycosides N-Acetyltransferase 30S ribosome Streptomycin Kanamycin

35 Antibiotics from Prokaryotes -- Macrolides Macrolide ring Sugars 50S ribosome Erythromycin Erythromycin

36 Antibiotics from Prokaryotes -- Tetracyclines 30S ribosome Tetracycline analog Tetracycline 7-Chlortetracycline (aureomycin) 5-Oxytetracycline (terramycin)

37 Antibiotics from Prokaryotes Daptomycin Lipid biosynthesis Cytoplasmic membranes Platensimycin

38 V. Antimicrobial Drug Resistance and Drug Discovery Antimicrobial Drug Resistance The Search for New Antimicrobial Drugs

39 Mechanisms of antibiotic resistance Horizontal Transfer in bacteria

40 26.12 Antimicrobial Drug Resistance PBPs Porin Β-lactamase Cell wall β-lactamase-catalyze hydrolysis of the β-lactam ring; Porin PBPs Cell wall Alteration of target site; Porin PBPs Cell wall Active transport out of cell (efflux pump).

41 26.12 Antimicrobial Drug Resistance Resistance Mechanisms The organism Lacks the target structure of the antibiotic Is impermeable to the antibiotic Alters the antibiotic Modifies the target of the antibiotic Develops a resistant pathway Pumps out the antibiotic

42 26.12 Antimicrobial Drug Resistance Phosphorylation Adenylation Streptomycin -Lactamase Penicillin Sites at which antibiotics are attacked by enzymes encoded by R plasmid genes Acetylation Chloramphenicol

43 26.12 Antimicrobial Drug Resistance

44 Percentage resistant fecal strains Gentamicin Nalidixic acid Chloramphenicol Ampicillin Sulfonamides Kanamycin Streptomycin Tetracycline Antimicrobial Drug Resistance The relationship between antibiotic use and the percentage of antibiotic resistant bacteria Antibiotic use (tons)

45 Percentage resistant strains of N. gonorrhoeae Antimicrobial Drug Resistance 9% 8% 7% 6% 5% 4% 3% 2% 1% Percentage of reported cases of gonorrhea caused by drug resistant strains 0% Year

46 Percentage resistant strains of N. gonorrhoeae Antimicrobial Drug Resistance Men who have sex with men Heterosexual men Women The prevalence of fluoroquinoloneresistant Neisseria gonorrhoeae 0 Massachusetts New York City General population

47 Candida albicans *? Key: Gram-negative Gram-positive Gram-positive/ acid-fast Fungus The appearance of antimicrobial drug resistance of some human pathogens Acinetobacter sp. Enterococcus faecium* Streptococcus pneumoniae Mycobacterium tuberculosis* Haemophilus ducreyi Salmonella typhi Haemophilus influenzae Neisseria gonorrhoeae *, some strains of these pathogens are now untreatable with known antimicrobial drugs. Pseudomonas aeruginosa* Salmonella sp. Shigella dysenteriae Shigella sp. Other gram-negative rods Staphylococcus aureus* Year

48 The temporary transfusion room was overcrowded

49 26.13 Search for NEW Antibiotics Strategies New analogs of existing antibiotics by 1) specific site modification and 2) by combinatorial chemistry Computer drug design Natural products

50 26.13 Search for NEW Antibiotics Vancomycin Modification at the position shown in red by substitution of a methylene (=CH 2 ) group for the carbonyl oxygen restores much of the lost activity

51 26.13 Search for NEW Antibiotics Saquinavir HIV protease Indinavir Computer-generated anti-hiv drugs

52 Other Means Better usage of existing antibiotics Drug combination antibiotics with chemical agents inhibit the resistance Other living organisms?

53 New medical term FMT:Fecal microbiota transplantation, reconstitution of normal flora by a stool transplant from a healthy individual has been shown to be effective in treating relapsing intestinal diseases Youngster I, et al.. JAMA, 2014

54 How does it work? Ecological placeholder of beneficial bacteria Engraftment resistance Immunomodulatory Decreased ph Nutrition of bacterial metabolites

55 The current research status Nature Reviews Microbiology, 2013, Gut microbiota and diseases Bacterial infections Obesity Colorectal cancer Type-2 diabetes Immunity diseases The human gut microbiota recovery from Vibrio cholerae infection. Subramanian S, et al. Nature, 2014 Role of the gut microbiota in immunity and inflammatory disease. Nature Reviews Immunology, 2013 Enrichment of the amounts of beneficial bacteria, such as Faecalibacterium spp during alleviation of type 2 diabetes with a Chinese herbal formula. Zhao L, et al. The ISME journal, Reduction of butyrate producers and increase of opportunistic pathogens may constitute a major structural imbalance of gut microbiota in colorectal cancer patients. Zhao L, et al. The ISME journal, 2011

56 Thanks for your attention!

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