Post on 01-Jan-2016
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Microbial Growth
Microbial growth implies an increase in cellular constituents
- leads to rise in cell number when microorganisms reproduce by processes like budding or binary fission
- ability to reproduce is a major criterion to determine if a microbe is alive or not - results when cell become longer/larger
Population growth is used to analyze the growth curve of a microbial culture
Cell Growth and Binary Fission
Fts proteins
forms in the space between the duplicated nucleoids
Min E proteins assistin the location of theactual cell midpoint
The FtsZ ringand cell division
forms a ring aroundthe cylinder in thecenter of the cell
2 copies of chromosomesare pulled apart to eachdaughter cell
Peptidoglycan Features
1. 3-D polymeric macromolecule
2. Formed from subunits by two types of covalent bonds
3. ß-1,4 glycosidic bonds between hexose sugars, and peptide bonds between amino acids
4. Determines cell shape and prevents osmolysis
5. Dynamic structure a) must grow as cell grows b) must be regulated to allow septation
PeptidoglycanPeptidoglycan
GA
MA
GA
MA
GA
MA
GA
MA
L-ala
D-glu
L-lys
D-ala
L-ala
D-glu
L-lys
D-ala
L-ala
D-glu
L-lys
D-alaL-ala
D-glu
L-lys
D-ala
-1,4-linkages
Glycanchains
Tetrapeptide
Peptide cross-link
Gram - negative Gram - positive
Peptidoglycan-Targeting AntibioticsPeptidoglycan-Targeting Antibiotics
• Destruction of peptidoglycan causes bacterial lysisDestruction of peptidoglycan causes bacterial lysis
• This can be accomplished in the laboratory using the This can be accomplished in the laboratory using the enzyme lysozyme, which hydrolyzes the glycosidic enzyme lysozyme, which hydrolyzes the glycosidic linkageslinkages
• Antibiotics should target bacteria-specific processes, Antibiotics should target bacteria-specific processes, such as peptidoglycan synthesissuch as peptidoglycan synthesis
• DO NOT use these antibiotics on bacteria with no cell wall DO NOT use these antibiotics on bacteria with no cell wall ((MycoplasmaMycoplasma)) or a cell wall that is not susceptible to or a cell wall that is not susceptible to them (them (MycobacteriaMycobacteria))
Antibiotics that Target the Antibiotics that Target the PeptidoglycanPeptidoglycan
• PhosphonomycinPhosphonomycin
• CycloserineCycloserine
• VancomycinVancomycin
• BacitracinBacitracin
• PenicillinPenicillin
• CephalosporinsCephalosporins
Peptidoglycan Antibiotic Targets
Synthesis-CytoplasmSynthesis-Cytoplasm
Start
NAG converted to NAM L-ala, D-glu and L-lys added one at a time
D-ala-D-ala added as a dipeptide
Synthesis-MembraneSynthesis-Membrane
Transfer to lipid carrier
Start
Addition of NAGresults in dipeptideprecursor
Sites of action of different antimicrobial agents. PABA, paraminobenzoic acid; DHFA, dihydrofolic acid; THFA, tetrahydrofolic acid.
Outer wall of Gram-positive and Gram-negative species and detail of porin channels of Gram-negative bacteria. Antimicrobial agents diffuse easily through the loose outer wall of Gram-positive bacteria, but must go through the narrow channels of the Gram-negative species.
Structure of metronidazole and its mechanism of action. Metronidazole enters an aerobic bacterium where, via the electron transport protein ferrodoxin, it is reduced. The drug then binds to DNA, and DNA breakage occurs.
Diagrammatic representation of inhibition sites of protein biosynthesis by various antibiotics that bind to the 30S and 50S ribosomes.
Inhibition of protein biosynthesis by aminoglycosides.
Structure of sulfonamide and trimethoprim with sites of inhibition of folic metabolism.
How do they do How do they do it?it?
• Acquire ability to degrade the Acquire ability to degrade the antibioticantibiotic
• Change their outer structure to Change their outer structure to prevent drug entryprevent drug entry
• Change the drug target so that it is no Change the drug target so that it is no longer affected by the druglonger affected by the drug
• Acquire and/or turn on an efflux pump Acquire and/or turn on an efflux pump to eliminate the drug from the cellto eliminate the drug from the cell
MULTIDRUG RESISTANCE AMONG PATHOGENIC BACTERIA
Where do they get Where do they get it?it?
• Chromosomal mutation(s) under Chromosomal mutation(s) under selective pressure by the antimicrobialselective pressure by the antimicrobial
• Conjugal transfer of resistance Conjugal transfer of resistance plasmidsplasmids
• Conjugal transfer of chromosomal Conjugal transfer of chromosomal resistance genesresistance genes
• Infection by bacteriophageInfection by bacteriophage
• An old system that found a new use An old system that found a new use (efflux pumps)(efflux pumps)
Example of how two antibiotics (A and B) may interact with synergy, indifference, or antagonism.
Types of Antibiotics
Penicillins have a common chemical structure which they share with the cephalopsorins. Penicillins are generally bactericidal, inhibiting formation of the cell wall.
•The natural penicillins are based on the original penicillin-G structure. Penicillin-G types are effective against gram-positive strains of streptococci, staphylococci, and some gram-negative bacteria such as meningococcus.
Types of penicillin
•Penicillinase-resistant penicillins, notably methicillin and oxacillin, are active even in the presence of the bacterial enzyme that inactivates most natural penicillins.
•Aminopenicillins such as ampicillin and amoxicillin have an extended spectrum of action compared with the natural penicillins. Extended spectrum penicillins are effective against a wider range of bacteria.
PenicillinPenicillin• Penicillin binds to proteins known as Penicillin-Binding Penicillin binds to proteins known as Penicillin-Binding
Proteins (PBPs)Proteins (PBPs)
• Multiple PBPs are made by each species, with different Multiple PBPs are made by each species, with different molecular weights and different enzymatic activitiesmolecular weights and different enzymatic activities
• PBPs are involved in the cross-linking reactions, and PBPs are involved in the cross-linking reactions, and typically have transpeptidase activitytypically have transpeptidase activity
• Inhibition of peptidoglycan cross-linking destabilizes the Inhibition of peptidoglycan cross-linking destabilizes the cell wallcell wall
Cephalosporins have a mechanism of action identical to that of the penicillins. However, the basic chemical structure of the penicillins and cephalosporins differs in other respects, resulting in some difference in the spectrum of antibacterial activity.
Like the penicillins, cephalosporins have a beta-lactam ring structure that interferes with synthesis of the bacterial cell wall and so are bactericidal. Cephalosporins are derived from cephalosporin C which is produced from Cephalosporium acremonium.
Cephalosporins
Tetracyclines got their name because they share a chemical structure that has four rings. They are derived from a species of Streptomyces bacteria.
Tetracycline antibiotics are broad-spectrum bacteriostatic agents, that inhibit bacterial protein synthesis. Tetracyclines may be effective against a wide variety of microorganisms, including rickettsia and amebic parasites.
Tetracycline
Structure of tetracycline showing the area critical for activity and major and minor points of modification.
The macrolide antibiotics are derived from Streptomyces bacteria, and got their name because they all have a macrocyclic lactone chemical structure.
The macrolides are bacteriostatic, binding with bacterial ribosomes to inhibit protein synthesis. Erythromycin, the prototype of this class, has a spectrum and use similar to penicillin.
The most commonly prescribed macrolide antibiotics are:erythromycin clarithromycin azithromycin dirithromycin roxithromycin
troleandomycin
Macrolides
Fluoroquinolones (fluoridated quinolones) are the newest class of antibiotics. Their generic name often contains the root "floxacin". They are synthetic antibiotics, and not derived from bacteria. Fluoroquinolones belong to the family of antibiotics called quinolones.
The older quinolones are not well absorbed and are used to treat mostly urinary tract infections. The newer fluroquinolones are broad-spectrum bacteriocidal drugs that are chemically unrelated to the penicillins or the cephaloprosins. Because of their excellent absorption fluroquinolones can be administered not only by intravenous but orally as well.
Fluoroquinolones
Aminoglycoside antibiotics are used to treat infections caused by gram-negative bacteria. Aminoglycosides may be used along with penicillins or cephalosporins to give a two-pronged attack on the bacteria.
This effect is bacteriocidal.
The most commonly-prescribed aminoglycosides:
amikacin gentamicin kanamycin neomycin streptomycin tobramycin
The aminoglycosides are drugs which stop bacteria from making proteins.
Aminoglycosides
- describe how the microbe grows in the fermenter- important to determine optimal batch times- growth of microbes can be broken down into 4 stages;
LAG PHASE LOG/EXPONENTIAL PHASE STATIONARY PHASE DEATH PHASE
MICROBIAL GROWTH KINETICS
Lag Phase
Cells have just been introduced into a new environment Cell growth is minimal Cell is synthesizing new components – no cell division takes place
- cell is old and depleted of ATP- medium may be different from the one the microorganism was growing- microorganism have been injured and require time to recover
accelerated growth phase
deceleratedgrowth phase
accelerateddeath phase
LOG/EXPONENTIAL PHASE
Cells have adjusted to their environmentRapid growth takes placeCell growth rate is highest in this phaseAt some point, cells growth rate level off and become constant
STATIONARY PHASE
Cell growth rate has leveled off and become constant Number of cells multiplying equals the number of cells dying
- nutrient limitation - aerobic organism are limited by oxygen availability - population growth cease due to the accumulation of toxic waste products
DEATH PHASE
Decline in the number of viable cells
Log/Exponential Phase
The rate of increase in biomass is correlated with the specificGrowth rate µ and the biomass concentration X (g/L), whereas theRate of sincrease in cell number is correlated with µ and cell density N (1/L)
d X = µ • X or d N = µ • N d t d t
specific growth rate, µ,
3 parameters : the concentration of limiting substrate S the maximum growth rate µmv
the substrate-specific constant Ks
µ = µm ___S___ Ks + S
Monod equationKs substrate concentration at which half the maximum specific growth rate is obtained (µ = 0.5 µm ) - equivalent to the Michaelis constant in enzyme kinetics
OrganismOrganism T T ººCC µµmm (h (h-1-1) Doubling time (h)) Doubling time (h)
Aspergillus nigerAspergillus niger 3030 0.20 3.46 0.20 3.46
Aspergillus nidulansAspergillus nidulans 2020 0.090 7.720.090 7.72
PenicilliumPenicillium 2525 0.123 5.650.123 5.65
Mucor hiemalisMucor hiemalis 2525 0.17 4.10.17 4.1
Fusarium avanaceumFusarium avanaceum 2525 0.18 3.80.18 3.8
Fusrium graminearumFusrium graminearum 3030 0.28 2.480.28 2.48
Verticillium agaricinumVerticillium agaricinum 2525 0.24 2.90.24 2.9
Geotrichum candidumGeotrichum candidum 2525 0.41 1.70.41 1.7
Neurospora sitophilaNeurospora sitophila 3030 0.40 1.730.40 1.73
Maximal specific growth rates (µm) of some fungi on glucose
Anderson et al, 1975
SubstrateSubstrate # of glucose units# of glucose units µµmm Doubling time Doubling time (h)(h)
GlucoseGlucose 11 0.280.28 2.482.48
MaltoseMaltose 22 0.220.22 3.153.15
MaltorioseMaltoriose 33 0.180.18 3.853.85
Anderson et al, 1975
Effect of glucose chain length on the maximal specific growth rate (µµmm)) in Fusarium graminearum at 30 ºC
PARAMETERS THAT MUST BE PRECISELY REGULATED
- temperature
- pH
- rate and nature of mixing
- oxygenation
- sterility and containment
Types of Cultures
1. Batch 2. Fed-batch 3. Continuous 4. Synchronous