Microbiology Zhenmei Lu ( 吕镇梅 ) lzhenmei@zju.edu.cn 2017 Spring-Summer
Chapter 26 Microbial Growth Control
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
I. Physical Antimicrobial Control 26.1 Heat Sterilization 26.2 Radiation Sterilization 26.3 Filter Sterilization
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
Survival fraction (log scale) 26.1 Heat Sterilization The effect of temperature over time on the viability of a mesophilic bacterium 100 10 1 70 C Decimal reduction time (D) 50 C 60 C 0.1 10 20 30 40 50 Time (min)
Decimal reduction time (min) 26.1 Heat Sterilization Relationship between temperature and the rate of killing in mesophiles and thermophiles 100 10 1 0.1 (a) (b) 100 105 110 115 120 125 130 Temperature ( C)
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
26.1 Heat Sterilization The Autoclave
Temperature ( C) 26.1 Heat Sterilization Autoclave time 130 Stop steam 120 110 Begin pressure Sterilization time 100 Flowing steam Temperature of object being sterilized Temperature of autoclave 0 10 20 30 40 50 60 Total cycle time (min) The moist heat sterilization Pasteurization
26.2 Radiation Sterilization Microwaves, UV, X-rays, gamma rays, and electrons can reduce microbial growth --UV A biological safety cabinet
26.2 Radiation Sterilization --Ionizing Radiation Electrons, hydroxyl radicals, and hydride radicals
Survival fraction (log scale) 26.2 Radiation Sterilization 1 0.1 D10 10% survival 0.01 Radiation (Grays) Relationship between the survival fraction and the radiation dose
26.2 Radiation Sterilization
26.3 Filter Sterilization A depth filter (HEPA) A Membrane filter A Nucleopore filter
26.3 Filter Sterilization
26.3 Filter Sterilization
II. Chemical Antimicrobial Control 26.4 Chemical Growth Control 26.5 Chemical Antimicrobial Agents for External Use
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
26.4 Chemical Growth control -- MIC by tube dilution technique
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 24 48 h Test organism shows susceptibility to some antibiotics, indicated by inhibition of bacterial growth around discs (zones of inhibition) after incubation
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
26.5 Chemical Antimicrobial Agents -- External Use
26.5 Chemical Antimicrobial Agents -- External Use
III. Antimicrobial Agents Used In Vivo 26.6 Synthetic Antimicrobial Drugs 26.7 Naturally Occurring Antimicrobial Drugs: Antibiotics 26.8 -Lactam Antibiotics: Penicillins and Cephalosporins 26.9 Antibiotics from Prokaryotes
Chemical Antimicrobial Agents -- In Vivo Use Animation: Antibiotic Modes of Action
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
Chemical Antimicrobial Agents -- In Vivo Use Annual worldwide production and use
Chemical Antimicrobial Agents -- Synthetic Antimicrobial Drugs Salvarsan one of the first useful antimicrobial agents Paul Ehrlich, 1900s
Chemical Antimicrobial Agents -- Synthetic Antimicrobial Drugs Sulfa drugs Sulfanilamide p-aminobenzoic acid Folic acid Gerhard Domagk, 1930s
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)
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
Antibiotics from Prokaryotes -- Aminoglycosides N-Acetyltransferase 30S ribosome Streptomycin Kanamycin
Antibiotics from Prokaryotes -- Macrolides Macrolide ring Sugars 50S ribosome Erythromycin Erythromycin
Antibiotics from Prokaryotes -- Tetracyclines 30S ribosome Tetracycline analog Tetracycline 7-Chlortetracycline (aureomycin) 5-Oxytetracycline (terramycin)
Antibiotics from Prokaryotes Daptomycin Lipid biosynthesis Cytoplasmic membranes Platensimycin
V. Antimicrobial Drug Resistance and Drug Discovery 26.12 Antimicrobial Drug Resistance 26.13 The Search for New Antimicrobial Drugs
Mechanisms of antibiotic resistance Horizontal Transfer in bacteria
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).
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
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
26.12 Antimicrobial Drug Resistance
Percentage resistant fecal strains Gentamicin Nalidixic acid Chloramphenicol Ampicillin Sulfonamides Kanamycin Streptomycin Tetracycline 26.12 Antimicrobial Drug Resistance 80 60 40 20 The relationship between antibiotic use and the percentage of antibiotic resistant bacteria 20 80 100 125 150 Antibiotic use (tons)
Percentage resistant strains of N. gonorrhoeae 26.12 Antimicrobial Drug Resistance 9% 8% 7% 6% 5% 4% 3% 2% 1% Percentage of reported cases of gonorrhea caused by drug resistant strains 0% 1981 1983 1985 1987 1989 1980 1982 1984 1986 1988 1990 Year
Percentage resistant strains of N. gonorrhoeae 26.12 Antimicrobial Drug Resistance 16 14 12 10 8 6 4 2 Men who have sex with men Heterosexual men Women The prevalence of fluoroquinoloneresistant Neisseria gonorrhoeae 0 Massachusetts New York City General population
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* 1950 1960 1970 1980 1990 2000 2010 Year
The temporary transfusion room was overcrowded
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
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
26.13 Search for NEW Antibiotics Saquinavir HIV protease Indinavir Computer-generated anti-hiv drugs
Other Means Better usage of existing antibiotics Drug combination antibiotics with chemical agents inhibit the resistance Other living organisms?
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
How does it work? Ecological placeholder of beneficial bacteria Engraftment resistance Immunomodulatory Decreased ph Nutrition of bacterial metabolites
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, 2014. 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
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