Population Growth Helmut Pospiech

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1 Population Growth Helmut Pospiech

2 Exponential Growth Since bacteria grow mainly by cell division, population growth can be described understood as increase in cell number: N = N 0 *2 n N: cell number N 0 : initial cell number n: generation number Brock Biology of Microorganisms, 13th ed.

3 Exponential Growth N = N 0 *2 n log N = log N 0 + n*log2 log N - log N 0 = n*log2 n = (log N - log N 0 )/log2 = (log N - log N 0 )/0.301 = 3.3*(log N - log N 0 ) Brock Biology of Microorganisms, 13th ed.

4 Exponential Growth Generation time g g = t/n t: time of culture Example: If you would start a culture with a single bacterial cell with a mass of appr g = 1 pg with a doubling time of 20 min (like E. coli), you would get after 1 day (24 h) 4.7 x g = 4700 tons, and after 2 days (48 h) 2.2 x g = 4000 x earth mass! microbial growth behaves commonly like a 1st order autocatalysed chemical reaction the growth rate constant k is defined as dn dt = kn form: or dx dt ln N - ln No k = t - to = kx respectively with, or in the integrated ln X - ln Xo t - to and k =, N: cell number X: cell mass t: time the generation time g is the average time from one cell division until the next g = ln2 k the doubling time td is the time needed to double the cell mass td = ln2 k

5 The Microbial Growth in static culture 1) Lag phase adjustment of the cells to the new medium Brock Biology of Microorganisms, 13th ed.

6 The Microbial Growth in static culture Brock Biology of Microorganisms, 13th ed. 2) Exponential growth phase cells grow at maximum growth rate until the first nutrient becomes limiting growth rate constant generation time and doubling time identical (cell size constant) since cell number and mass increase exponentially, exponential growth occurs only for a short period, because resources for growth are limiting

7 The Microbial Growth in static culture 3) Stationary phase nutrients become exhausted or toxic metabolites accumulate, the rate of growth declines and growth eventually stops cells in stationary phase are generally smaller than in exponential phase (cell division stops later than cell mass production) stationary phase cells are more resistent to physical and chemical agents stationary cells often produce secondary metabolites like antibiotics Brock Biology of Microorganisms, 13th ed.

8 The Microbial Growth in static culture 4) Death phase bacterial cells in non-growing stage eventually die depletion of the cellular reserves of energy death occurs as growth in an exponential rate the death rate is very variable depending on the particular organism as well as on the environmental conditions Brock Biology of Microorganisms, 13th ed.

9 Continuous Culture the Chemostat Continuous addition of fresh medium and equal amount of culture Brock Biology of Microorganisms, 13th ed.

10 Measurement of Bacterial Growth 1. Direct Methods 1.1 Measurement of Cell Number a) counting cells in the microscope - counting chamber special microscope slide (have a defined volume between slide and cover slip total cell count but vital staining (e.g. with methylene blue which is efficiently excluded from yeast cells) may allow viable cell count Brock Biology of Microorganisms, 13th ed.

11 Measurement of Bacterial Growth 1. Direct Methods 1.1 Measurement of Cell Number a) counting cells in the microscope - counting chamber special microscope slide (have a defined volume between slide and cover slip total cell count but vital staining (e.g. with methylene blue which is efficiently excluded from yeast cells) may allow viable cell count b) Coulter counter automatic method often used in medicine (e.g. count of blood cells)

12 Measurement of Bacterial Growth 1. Direct Methods 1.1 Measurement of Cell Number c) plate count viable cell count very reliable, but may cause problems when a large quantity of samples has to be processed at the same time Brock Biology of Microorganisms, 13th ed.

13 Measurement of Bacterial Growth 1. Direct Methods 1.1 Measurement of Cell Number c) plate count viable cell count very reliable, but may cause problems when a large quantity of samples has to be processed at the same time Brock Biology of Microorganisms, 13th ed.

14 Measurement of Bacterial Growth 1.2 Measurement of Cell Mass no differentiation between living and dead cells can be made a) fresh weight mass of the cells from a defined volume after filtering, centrifugation etc. quick and simple, but very unreliable because of the variation in water content b) dry weight mass of the cells from a defined volume after separation from the medium and drying more work, but better data

15 Measurement of Bacterial Growth 2. Indirect Methods 2.1 Spectrometric Determination measurement of the optical density of the cell suspension commonly a wavelength of l = 600 nm is used linear relationship between cell number and optical density only for OD < appr. 0.6! probably most commonly used Brock Biology of Microorganisms, 13th ed.

16 Measurement of Bacterial Growth 2. Indirect Methods 2.1 Spectrometric Determination measurement of the absorbance of the cell suspension commonly a wavelength of l = 600 nm is used linear relationship between cell number and absorbance only for OD < appr. 0.6! probably most commonly used 2.2 Measurement of Cellular Constituents protein, nucleic acids, ATP etc. indirect methods can only give estimations of the cell number and mass unless being calibrated with a direct method for a specific application

17 Control of Microbial Growth Some definitions: Sterilisation the killing or removal of all viable organisms (from a growth medium, instrument or surface) Decontamination the treatment of an object or surface to make it safe to handle Disinfection the killing or removal of pathogenic organisms Preservation treatment to limit growth of (micro-) organisms for a transient time (e.g. to extend the shelf life of food stuff) Antibiosis the killing or limitation of growth of microbial pathogens within an other organism (human, cattle, plants etc.) Brock Biology of Microorganisms, 13th ed.

18 Physical Antimicrobial Control Heat sterilisation Most vegetative cells of mesophilic bacteria will die at temperatures above 70 C Denaturation of enzymes and nucleic acids Disruption of membranes Endospores are most heat resistant Geobacillus stearothermophilus endospores are among the most heat-resistant forms of life and are used to control sterilisation efficiency The decimal reduction time D is the time required to kill 90% of the cells of a certain microorganism Brock Biology of Microorganisms, 13th ed.

19 Heat sterilisation Dry heat Glass ware and other heat resistant equipment Microorganisms are much more resistant to dry compared to moist heat 2 hours at 160 C or 1 hour at 180 C Moist heat (autoclavation) Media, glass ware etc. Most commonly used method In automatic or semi-automatic instruments In water steam in the absence of air at 2.1 atm (2.06 bar) pressure / 121 C Pasteurisation Partial killing ( desinfection ) of foodstuff to remove pathogens and prolong shelf life with minimal effect on nutritional value and appearance Kills pathogens such as Mycobacterium bovis/tuberculosis, Brucella spp., Salmonella, Listeria monocytogenes, Campylobacter spp., Escherichia coli e.g. 65 C for 30 minutes (bulk pasteurisation) or 71 C for 15s (flash pasteurisation)

20 Autoclave Brock Biology of Microorganisms, 13th ed.

21 Radiation Treatment Ultraviolet Radiation Wavelength nm (254 nm) Destroys mainly DNA and thereby kills the organism Used for surface and water sterilisation Low penetration of most materials (except clear liquids) Brock Biology of Microorganisms, 13th ed.

22 Radiation Treatment Ionising Radiation (IR) High energy electromagnetic radiation (e.g. Roentgen rays) Destroys mainly nucleic acids and proteins and thereby kills the organism Variable resistance Also destroys nutrient without changing the outer appearance of food stuffs Brock Biology of Microorganisms, 13th ed.

23 Radiation Practices IR sources: Cathode electron beams X-ray machines Nuclides such as 60 Co or 137 Cs Good penetration of solids and liquids except metals Use for food sterilisation and decontamination approved, but not generally accepted Brock Biology of Microorganisms, 13th ed.

24 Filter Sterilisation Depth filter Fibrous sheet Borosilicate (glass) fibre HEPA (high-efficiency particulate air) filter Mainly air filters Membrane filters Cellulose acetate, cellulose nitrate or polysulfone High tensile strength Contain large numbers of small holes (pores) Pore size can be controlled during manufacturing process) <0.2 µm pore size is required for sterilisation Viruses, mycoplasms and some spirlliae will pass through 0.2 µm filters Micropore filters are produced of thin (10 µm) Polycarbonate by radiation followed by chemical etching Brock Biology of Microorganisms, 13th ed.

25 Filter Sterilisation Brock Biology of Microorganisms, 13th ed.

26 Chemical Antimicrobial Control Antimicrobial component Natural or synthetic chemical that kills or inhibitd growth of an microorganism -cidal indicates an agent that kills the microorganism -static indicates an agent that does not kill but inhibits growth of an microorganism (e.g. bacteriostatic, fungizidal) Different effects Bacteriostatic Bacteriozidal Bacteriolytic Brock Biology of Microorganisms, 13th ed.

27 Measuring the Antimicrobial Activity The tube dilution technique is used to determine the Minimum Inhibitory Concentration of an antimicrobial agent in respect of a certain microorganism The disc diffusion technique is used to determine the Susceptibility spectrum of a certain microorganism against several antimicrobial agent

28 Chemical Antimicrobial Agents for External Use Sterilants Destroy all microbial life Mainly used for materials that cannot be autoclaved or irradiated Ethylene oxide Formaldehyde Peroxyacetic acid Hydrogen peroxide Hypochlorite (bleach) Amylphenol Disinfectants Kill microorganisms, but not necessarily endospores Used on inanimate objects (e.g. floors, tables etc.) Ethanol Cationic detergents Sanitizers Reduce microbial numbers, but do not eliminate them completely E.g. for instruments used in food chemistry Chloramine Hypochlorite Antiseptics and Germicides Kill or inhibit growth of microorganisms Can be applied to living tissue Ethanol Iodine Hydrogen peroxide

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31 Antimicrobial Agents Used In Vivo Compounds with selective toxicity: Pathogen is more sensitive than the host The greater the difference in sensitivity, the more of the agent can be applied - off-target effects! Search for target (e.g. essential metabolic feature of the pathogen that is not present in the host Synthetic Antimicrobial Drugs Naturally Occuring Antimicrobial Drugs: Antibiotics Natural compound produced by a microorganism with antimicrobial activity against other microorganisms Mainly secondary metabolites Producer always has natural resistance against the antibiotic produced Mainly by Steptomycetes and related Gram-positive bacteria

32 A magic bullet against Paul Ehrlich Salvasan to treat syphilis Scientific break-through But severe side-effects Difficult to administer because of low solubility microorganisms? Around 100 years ago, German chemists and physicians experimented with with dyes (e.g. azodyes and metal-organics to find compounds that selectively kills bacteria and could be used in treatment of infectious disease Gerhard Domagk Sulfonamide (Sulfa drugs) The principle of using growth factor analogues as synthetic drugs was born Resistance require only one point mutation Still used in combination with another folic acid synthesis inhibitor, trimethoprim

33 Targets of Antimicrobial agents

34 Other growth factor analogues P-fluorphenolalanine Amino acid analogue Nucleoside analogues Blocking nucleic acid synthesis Mainly used in the treatment of viral and fungal infections (and cancer) Isoniazid Nicotinamide analogue Interferes with mycolic acid synthesis in Mycobacterium Used to treat tuberculosis

35 Classes of Antibacterial Drugs Quinolones Inhibit gyrase, a topoisomerase only present in bacteria Several quinolones are now in heavy use against several bacterial diseases, e.g. Tuberculosis Anthrax

36 Penicillins The first antibiotics discovered by Alexander Fleming by chance in 1929 Produced by the mold Penicillium chrysogenum Inhibitor of transpeptidase (bacterial cell wall synthesis) Now only semi-synthetic penicillins are used that can be orally administered and show improved spectrum against different bacteria or evasion of resistance

37 By molds of the genus Cephalosporium Also β-lactam antibiotics Same mode of action as penicillin Mainly semi-synthetic cephalosporins used Cephalosporin has to side chains that can be modified, penicillin only one More possibilities to apply combinatorial chemistry Testing systematically or structureguided combinations of new side chains to obtain new derivatives with improve properties, e.g. - Improved activity - Broader spectrum - Better administration Cephalosporins

38 E.g. Streptomycin (Streptomyces griseus), kanamycin, gentamycin, neomycin, spectinomycin etc. Target 30S subunit of the ribosome Inhibit protein synthesis Useful against Gramnegative bacteria Serious side effects (e.g. nephrotoxicity) Aminoglycosides

39 Lacton rings bonded to sugars E.g. erythromycin (Streptomyces erithreus) Targets the 50S subunit of the ribosome Inhibits protein synthesis Broad spectrum antibiotics Particularly use against legionellosis and for the treatment of small children Macrolides

40 By Streptomyces species Target 30S subunit of the ribosome Inhibit protein synthesis Both natural and semisynthetic tetracyclins in use Second most commonly used antibiotic Overuse in non-medical applications (e.g. farming) causes increasing occurence of resistance Tetracyclin

41 Daptomycin Novel antibiotic with new mode of action By Streptomyces species Cyclic peptide Forms pores in the bacterial membrane Loss of membrane potential

42 Platensimycin Completely new class of antibiotic by Streptomyces platensis Disrupts bacterial lipid synthesis Potential new last line of defence antibiotic

43 Vancomycin Inhibits cell wall synthesis After occurance of resistance, a modified Vancomycin has been developed that evades resistance

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46 Types of resistance

47 The appearance of antimicrobial drug resistance

48 Types of resistance

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50 Antifungal agents

51 Antifungal agents

52 How to find new antibiotics Systematic screening Mainly soil samples around the world It was said that the researcher from pharma companies broad along soil sample from all over the world whenever they visited a new place! Brock Biology of Microorganisms, 13th ed.

53 Discovery void Silver, Clin. Microbiol. Rev. 2011, 24(1):71.

54 Current status in AB development Cooper and Shlaes, Nature 2011, 472, 32

55 Enrichment and Selection Inoculum is the material from which an microorganism is isolated, e.g. Soil Transformation of a cloning experiment Sample from a diseased patient Food sample from a restaurant after a customer got gastroenteritis What if the bacteria of interest represent only a small fraction of all the microorganisms present? Brock Biology of Microorganisms, 13th ed.

56 Enrichment and Selection Creation of conditions that favour the microorganisms of interest Omit nutrients of growth factors that other (contaminating) microorganisms require, e.g. No nitrogen source for nitrogen-fixing bacteria Add chemicals that prevent the growth of other organisms, e.g. Ampicillin in culture medium to select for E. coli that have uptaken a plasmid with an ampicillin resistance gene Choose sample preparation of growth conditions that favour the microorganisms of interest or harm/kill the unwanted microorganisms, e.g. Boiling of the inoculum to select for endospore formers (all vegetative cells will die) Incubation at 37 C for human pathogenic bacteria ph 4-5 for lactic acid bacteria or yeasts

57 Inoculum Enrichment Selection Pure Culture Brock Biology of Microorganisms, 13th ed.