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