Bacterial Growth &

Metabolism

Dr. Aishvarya HajareIst year PostgraduateDept of Periodontics

Contents Introduction Bacterial growth1. Bacterial growth curve2. Kinetics of microbial growth3. Bacterial growth in vivo4. Factors affecting microbial growthBacterial metabolism

1. Components of metabolism 2. Role of ATP 3. Metabolic Pathways of energy generation

Bacterial flora in oral cavityA. actinomycetocomitans and P. gingivalisConclusion References

Introduction

Life on Earth is famous for its diversity.

Throughout the world we can find many millions of different forms of life.

Biologic classification helps identify each form according to common properties using a set of rules and an estimate as to how closely related it is to a common ancestor in a way to create an order.

Introduction

Eukaryote, any cell or organism that possesses a clearly defined nucleus.

Has a nuclear membrane that surrounds the nucleus, in which the well-defined chromosomes are located.

also contain organelles, including mitochondria, Golgi apparatus, endoplasmic reticulum and lysosomes.

- “Encyclopædia Britannica”

Prokaryote is any organism that lacks a distinct nucleus and other organelles due to the absence of internal membranes.

The cell membrane is made up of phospholipids and constitutes the cell’s primary osmotic barrier.

The cytoplasm contains ribosomes, which carry out protein synthesis, and a double-stranded deoxyribonucleic acid (DNA) chromosome, which is usually circular.

- “Encyclopædia Britannica”

Classifying Microbes According to Their Energy and Carbon Sources.

Based on energy source Phototrophs• Use light as an energy source; photosynthesize. Chemotrophs• Use inorganic and organic chemicals.

Based on carbon source Autotrophs• Use carbon dioxide. Heterotrophs• Do not use carbon dioxide as their carbon source

Bacterial growth Cells are the most fundamental units of life. All living organisms are made of one or more. Cells reproduce by copying their genetic material and then dividing

—a parent cell giving rise to daughter cells.

Types of Cell Division: 1) Binary Fission 2) Mitosis & Meiosis

It refers to an increase in cell numbers, not in cell size.

Binary Fission The reproduction of prokaryotic

cells (bacteria and bacteria-like Archaea) is accomplished through binary fission.

A bacterial cell that is ready to divide first copies its genetic material, called the nucleoid—a single, circular chromosome of DNA (deoxyribonucleic acid).

The two chromosomes, each attached to the plasma membrane, move apart as the cell elongates.

Once the two copies of genetic instructions are separated, the cell divides, laying down new cell wall and membrane between the two chromosomes.

Binary fission is essentially cloning.

Barring mutations that may have occurred when the genetic material was copied (a process called replication), the two resulting daughter cells are identical to the parent cell.

Alternative means

Budding

Fragmentation

Conidiospores (filamentous bacteria)

Bacterial growth curve Illustrates the dynamics of growth

Phases of growth

1. Lag phase

2. Exponential or logarithmic (log) phase

3. 4. Stationary phase

5. Death phase (decline phase)

LAG PHASE During this phase, bacteria are growing in size, but they are not

undergoing binary fission.

Hence, there is no increase in cell number.

The bacteria are adapting to the new environment and are synthesizing cellular components such as ribosomes, enzymes, and other proteins.

Bacteria have the maximum cell size towards the end of the lag phase.

LOG PHASE This phase is also referred to as the exponential phase because

there is a logarithmic increase in cell number.

This exponential growth is expressed as the bacteria’s generation time.

During this phase, the conditions are optimal for growth and binary fission occurs.

In the log phase, cells are smaller and stain uniformly.

STATIONARY PHASE There is no net increase or decrease in cell number in this stage. In

other words, cell growth (division) equals cell death.

The birth rate decreases due to limited nutrients, lack of space, and the build up of secondary metabolic products (e.g. toxins).

The insufficient supply of nutrients also causes some bacteria to form spores during this phase.

Cells frequently are gram variable and show irregular staining due to the presence of intracellular storage granules.

PHASE OF DECLINE This phase is characterized by an exponential death of cells.

When the media runs out of nutrients and there are too many toxins, cells begin to die at a faster rate.

Involution forms are common in the phase of decline.

FACTORS AFFECTING GROWTH

1. Water

2. Oxygen

3. Carbon dioxide

4. Temperature

5. Hydrogen ion concentration

6. Light

7. Osmotic pressure

8. Symbiosis and antagonism

WATER Moisture is essential for the life of bacteria. Most processes taking

place in a bacterial cell are in a water base.

80% of bacterial cell consists of water.

Dehydration is detrimental for most bacteria eg. Treponema pallidum. But some like staphylococcus can resist drying for months.

Spores are particularly resistant to dessication and may survive in the dry state for several decades.

OXYGEN Most life forms depend on oxygen for survival & growth.

Microrganisms require oxygen to act as terminal electron acceptor in their respiratory chain.

AEROBE = bacteria which require oxygen for growth.

ANAEROBES = bacteria which do not require oxygen for growth.

OBLIGATE AEROBE = bacteria which grow only in the presence of oxygen. Eg. Pseudomonas, bacillus.

FACULTATIVE ANAEROBE = bacteria which are aerobes but can grow with lack of oxygen or in absence of oxygen. Eg. Streptococci

MICROAEROPHILIC = bacteria which grow with trace amount of oxygen. Eg. Listeria monocytogenes

OBLIGATE ANAEROBES = bacteria which strictly grow in the absence of oxygen. They may die on exposure to oxygen. Eg clostridia, bacteroides.

AEROTOLERANT ANAEROBES = anaerobes which do not require oxygen but tolerate the presence of oxygen.

CARBON DIOXIDE Approximately half of dry weight

CO2 is provided by cellular metabolism and from environment.

Autotrophic organisms are able to use carbon dioxide as source of carbon.

Heterotrophic bacteria require some amount of carbon dioxide from exogenous sources.

5-10 % CO2 is supplied for them in culture.

Capnophilic = requiring excess amount of CO2 eg Brucella abortus (10% CO2).

The carbon available in the carbohydrate sugar molecules is cycled further by microorganisms in a series of reactions that tricarboxylic acid (or TCA) cycle.

The breakdown of the carbohydrate serves to supply energy to the microorganism.

This process is also known as respiration.

In anaerobic environments, microorganisms can cycle the carbon compounds to yield energy in a process known as fermentation

TEMPERATURE PSYCHROPHILES = bacteria which grow below 20°C, e.g. soil and

water saprophytes. Up to -7°C reported.

MESOPHILES = bacteria which grow between 20-40°C. e.g.. Most pathogenic bacteria are mesophiles. Wide range e.g. Pseudomonas 5-43°C, narrow range e.g. Gonococcus 30-39°C.

THERMOPHILES = bacteria which grow at higher temperature i.e. 60-80°C. e.g. Bacillus stearothermophilus. Up to 250°C reported.

THERMAL DEATH POINT = The lowest temperature that kills a bacterium under standard conditions in a given time.

Under moist conditions most vegetative, mesophilic bacteria have a thermal death point 50 to 65°C and most spores between 100 and 120°C

HYDROGEN ION CONCENTRATION

The pH requirement of bacteria is also variable.

Most bacteria have an average pH requirement of 7.2-7.6 which matches with pH in human body environment.

Some bacteria grow in acidic pH. Eg lactobacilli in pH=3.

Some bacteria grow in alkaline pH. Eg. Alkaligenes at pH = 10.5

LIGHT Most bacteria prefer darkness for growth.

Cultures die if exposed to sunlight.

However some bacteria require sunlight and are called phototropic.

Exposure to light may influence pigment production.

Photochromogenic mycobacteria form a pigment only on exposure to light and not when incubated in the dark

OSMOTIC PRESSURE There is a wide range of osmotic tolerance found in bacteria.

0.5% NaCl is added in culture media to provide suitable osmolarity.

PLASMOLYSIS = Sudden exposure to hypertonic solutions may cause osmotic withdrawal of water and shrinkage of protoplasm. This occurs more readily in gram negative than in gram positive bacteria.

PLASMOPTYSIS = Sudden transfer from a concentrated solution to distilled water may cause plasmoptysis (excessive osmotic imbibition leading to swelling and rupture of the cell).

Nitrogen: Found in all the amino acids, nitrogenous bases of nucleic acids, etc.

Hydrogen: found in all biological molecules, Carbs, fats, proteins, nucleic acids, etc

Phosphorous: found in nucleic acids, ATP, and phospholipdids of membranes

Sulfur: found in 2 or 3 amino acids of microbes

Trace elements: inorganic elements needed in very tiny concentrations (manganese, cobalt, Zn, Cr)

s

Generation time : The time required for a bacterium to give rise to two daughter cells under optimum conditions is known as generation time.

Kinetics of microbial growth : unicellular organisms divide by binary fission each cell grows to full size, replicates its genetic material then divides into identical daughter cells , leading to exponential increase in cell numbers.

1 2 4 8 2 n

MAINTENANCE OF CELLS IN EXPONENTIAL PHASE (CONTINUOUS CULTURE)

This is done by repeatedly transferring bacterial cells into fresh medium of identical composition.

Transfer is done while they are multiplying in exponential phase.

Two techniques are used: Chemostat device Turbidostat device

Maintenance of bacteria in continuous culture is sometimes necessary in industrial and research purposes.

Bacterial growth in vivo In vitro and in vivo growth patterns of bacteria are different as the

environmental conditions are different.

In vivo growth of bacteria depends on our nutritional status, immunity, hormonal factors, pH, redox potential etc.

There is a significant difference in the growth patterns of bacteria in human body and artificial medium.

Relationship Involving Microorganisms

Despite an apparent simplicity, bacteria can form complex associations with other organisms. This process is known as symbiosis.

Symbiosis - defined as the living together in more or less intimate association or close union of two dissimilar organisms

The organisms that live together in such relationship are called Symbionts. 

4 types : MUTALISM COMMENSALISM PARASITISM SYNERISIM

Mutalism A type of symbiotic interaction in which both organisms benefit from

the relationship in some way.

In humans, gut bacteria assist in breaking down additional carbohydrates, out-competing harmful bacteria, and producing hormones to direct fat storage.

The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, and preventing colonization of the intestine with pathogenic bacteria

Commensalism one organism benefits while the other organism neither benefits nor

suffers from the interaction.

Humans are host to a variety of commensal bacteria in their bodies that do not harm them but rely on them for survival.

e.g. staph epidermis on skin

Parasitism Parasitic relationships, in which one species benefits and the other

suffers, are very common in nature.

Most of the microorganisms studied in medical microbiology are parasitic and feed on human tissue.

For e.g., cholera, leshmaniasis, and Giardia are all parasitic microbes.

BACTERIAL METABOLISM METABOLISM = the series of changes of a substance

(carbohydrate, protein, fat) that take place within the bacterial cell from absorption to elimination is known as metabolism.

CATABOLISM = breakdown of macromolecules into simpler micromolecules, absorption into cell, conversion into basic blocks including interconversion of ADP to ATP.

ANABOLISM = a process by which the basic building blocks are utilized in synthesis of various cellular structures such as monomers and polymers.

Aerobic bacteria obtain their energy and intermediates only through OXIDATION and energy is provided by ATP (oxidative phosphorylation).

Anaerobic bacteria obtain their energy by FERMENTATION (substrate level phosphorylation).

Facultative anaerobes may act in both ways.

Glucose is a key energy-storing molecule

Nearly all cells metabolize glucose for energy

Glucose metabolism is fairly simple

Other organic molecules are converted to glucose for energy harvesting

Components of metabolism Enzymes

Adenosine triphosphate (ATP)

Energy source

Electron carriers

Precursor metabolites

biological catalyst, facilitates each step of metabolic reaction by lowering the activation of energy reaction.

Serves as energy currency of cell

Compound that is oxidized to release energy, also called as electron donor.

Carry the electrons that are removed during oxidation of energy source.

Intermediate metabolite that link anabolic & catabolic pathway.

Components Functions

Overview of Glucose Breakdown The overall equation for the complete breakdown of glucose is:

C6H12O6 + 6O2 6CO2 + 6H2O + ATP

The main stages of glucose metabolism are:o Glycolysiso Cellular respiration

Overview of Glucose Breakdown - Glycolysis

Glycolysis

Occurs in the cytosol

Does not require oxygen

Breaks glucose into pyruvate

Yields two molecules of ATP per molecule of glucose

If oxygen is absent fermentation occurs

Pyruvate is converted into either lactate, or into ethanol and CO2

If oxygen is present cellular respiration occurs

Pathways alternative to glycolysis Many bacteria have another pathway in addition to glycolysis for

degradation of glucose.

1. Pentose Phosphate Pathway

2. Entner Doudoroff Pathway

Pentose phosphate pathway Hexose monophosphate shunt Occurs simultaneously with glycolysis & provide breakdown of both

pentose sugar and glucose. Intermediate pentoses are used for nucleic acid synthesis,

aminoacid synthesis Important producer of reduced coenzyme i.e. NADPH used for

biosynthetic reaction.

Stuart Hogg Essential microbiology 2nd edition John Wiley & Sons.Ltd 2005

Entner-Doudoroff pathway

Uses 6-phosphogluconate dehydratase and 2-keto-3-

deoxyphosphogluconate aldolase to create pyruvate from glucose. Most of gram –ve bacteria like pseudomonas, rhizobium,

agrobacterium. Produces 1 molecule NADH, 1 molecule NADPH and 1 molecule of

ATP

Stuart Hogg Essential microbiology 2nd edition John Wiley & Sons.Ltd 2005

Overview of Glucose Breakdown- Cellular Respiration

Cellular respiration - pyruvate obtained from glucose breakdown are channeled either to respiration or fermentation.

Requires oxygen

Breaks down pyruvate into carbon dioxide and water

Reactions in Mitochondrial Matrix (Eukaryotes) or Cytoplasm

(Prokaryotes)

Electron Transport Chain

Last phase of respiration which generate ATP from reduced

substrates. Consists of a sequence of carrier molecules through which electron

passes. Occurs in plasma membrane. Electron transport chain is different in different bacteria.

Electron Transport Chain

Fermentation

Fermentation is the process of extracting energy from the oxidation

of organic compounds, such as carbohydrates, using an

endogenous electron acceptor, which is usually an organic

compound. In contrast, respiration is where electrons are donated to an

exogenous electron acceptor, such as oxygen, via an electron

transport chain.

Anaerobic respiration

Microbiology of oral cavity The colonization of the oral cavity starts close to the time of birth.

Within hours after birth, the sterile oral cavity will be colonized by low numbers of mainly facultative and aerobic bacteria.

It is estimated that more than 700 different species are capable of colonizing the adult mouth and that any individual typically harbours 150 or more different species. (Moore WE, 1994)

Most oral bacteria are harmless commensals under normal circumstances.

The process begins with the colonization of habitat by pioneer microbial populations.

In oral cavity of newborns, streptococci are the pioneer organisms.

They fill the niche of the new environment and modify the habitat and new population develops.

Ecosystems of oral cavity

1. Buccal epithelium (soft tissue)

2. Dorsum of the tongue

3. Supragingival tooth surface

4. Subgingival tooth and crevicular epithelial surfaces

Complexity of oral flora

Biofilm is an association of micro-organisms in which microbial cells

adhere to each other on a living or non-living surfaces within a self-

produced matrix of extracellular polymeric substance.

Biofilm formation is a multi-step process starting with attachment to a

surface then formation of micro-colony that leads to the formation of

three dimensional structure and finally ending with maturation

followed by detachment.

The dental plaque biofilm also has similar structure.

The term ‘Biofilm’ was coined by Bill Costerton in 1978.

The term Biofilm (Wilderer and Charaklis 1989) describes the relatively

indefinable microbial community associated with a tooth surface or any

other hard non-shedding material, randomly distributed in a shaped

matrix or glycocalyx.

Biofilm is “a microbially derived sessile community characterized by cells

that are irreversibly attached to a substratum or interface or to each

other, embedded in a matrix of extracellular polymeric substances that

they have produced, and exhibit an altered phenotype with respect to

growth rate and gene transcription.” Donlan and Costerton 2002

Dental plaque has been defined as “a specific but highly variable

structural entity consisting of micro-organisms and their products

embedded in a highly organized intercellular matrix.”

It represents a true biofilm consisting of a variety of micro-organisms

involved in a wide range of physical, metabolic and molecular

interactions.

Lang NP, Mombelli A, Attstrom R. Oral Biofilms and Calculus. In: Lindhe J, Lang NP, Karring T, editors. Clinical Periodontology and Implant Dentistry. 5th ed. Oxford: Blackswell- Munksgaard; 2008. pp. 183–267.

Dental plaque (WHO 1978) Is defined as specific but highly variable structural entity resulting

from sequential colonization & growth of microorganisms on the

surfaces of teeth & restoration consisting of microrganisms of

various strains and species embedded in the extracellular matrix,

composed of bacterial metabolic products & substances from

serum, saliva & blood.

Actinobacillus actinomycetecomitans

The term Actinobacillus actinomycetemcomitans was coined by

Topley and Wilson.

Member - Actinobacillus Family – Pasturellaceae Given the name Actinobacillus actinomycetemcomitans, that is,

derived from the Greek words, actes- meaning ray , because of the

star on the top of the agar colony and, Mycetes meaning fungus,

because Actinomyces was originally thought to be a fungus. The Latin word Comitans, meaning in common with, or

accompanying Actinomycetes spp.

Studies found similarity of A. actinomycetemcomitans and Haemophilus aphrophilus, H. paraphrophilus, and H. segnis, suggesting the new genus Aggregatibacter for them.

The species of the genus Aggregatibacter are independent of X factor and variably dependent on V factor for growth in vitro.(Nørskov-Lauritsen N; Kilian M, 2006)

Related A.a to Juvenile periodontitis (Newman, Socransky and Slots 1976)

General characteristics of A. Actinomycetemcomitans

Fastidious Facultatively anaerobic Non-motile Non-sporing Non-hemolytic Small gram-negative rod, 0.4–0.5 lm x 1.0–1.5 lm in size Oxidase +ve and Catalase +ve

Colony characteristics Blood agar or chocolate blood agar supplemented with 5–7%

defibrinated horse blood if incubated in an aerobic atmosphere supplemented with 5– 10% carbon dioxide.

are generally round with an irregular edge, domed and colourless in appearance, and may have central wrinkling and adhere to the agar surface.

The pale colour of the agar allows light to be transmitted through the agar, and lighting the agar plate from underneath allows easy discrimination of the internal structures.

Adherent, catalase-positive colonies, with star-like internal structures, that reduce nitrates to nitrites are characteristic of A. actinomycetemcomitans

Porphyromonas Gingivalis

belongs to the phylum Bacteroidetes is a nonmotile, Gram-negative, rod-shaped, anaerobic,

pathogenic bacterium. It forms black colonies on blood agar. has been considered as one of the putative periodontal pathogen. Colonizes periodontal pocket and spreads into deeper tissues,

including connective tissue and bone. P.gingivalis is frequently isolated from subgingival plaque samples in

patients with adult and other forms of periodontitis.

Major oral ecologic niche for this species appears to be subgingival

plaque Possess significant proteolytic activity. P.g is at present the only known porphyromonas species isolated

from human that produces phenyl acetic acid as a metabolic end

product.

Asaccharolytic and black pigmented rod that form greenish-black

colonies in blood agar plates (Haffajee and Socransky, 1994).

Fresh clinical isolates of this organism have different colony

morphologies, ranging from smooth to rough colony morphotypes

(Reynolds et al., 1989).

Tannerella forsythia

The original isolate, identified as a “fusiform Bacteroides,” was

first reported in the literature by Tanner et al. in 1979.

Is an non motile,

spindle-shaped,

highly pleomorphic rod and

gram negative obligate anerobe

Colony characteristics

It grows slowly only on anerobic conditions

Needs several growth factors such as N-acetylmuramic acid

Produces several proteolytic enzymes that are able to destroy

immunoglobulins and factors of complement system.

Prevotella intermediaand

Prevotella nigrescens

Species from this group are

Short Round-ended

Non motile

Gram negative

Colony characteristics Grow anaerobically, with dark pigmenation on blood agar.

Campylobacter rectus C. rectus is a Gram negative, anaerobic, short, motile vibrio.

The organism is unusual in that it utilizes H2 or formate as its energy source.

Grows anaerobically with dark pigmentation when sulfide is added to the medium, which is transformed to FeS, giving a gray stain

Conclusion

Getting well acquainted with the bacterial growth &

metabolism helps to

Discrimate between the various growth patterns of

microorganisms. Association of microorganisms with specific disease. Determine the treatment plan necessary for the disease.

References Stuart Hogg Essential microbiology 2nd edition John Wiley & Sons.Ltd

2005

Alternatives to binary fission in bacteria. E. R. Angert. Nature Reviews Microbiology (2005) vol. 3, pp. 214-224

Newman MG,Takkei HH, Klokkevold PR, Caranza FAJr. Caranza’s Clinical Periodontology 10th edition St Louis. Missisouri Saunders Elseveir Inc 2006:s

Audesirk, Audesirk & Byers, Biology: Life on Earth 10 th edition Pearson Higher Ltd 2013

Pudlo NA, Urs k, Kumar SS, German JB, Mills DA, Martens EC Symbiotic Human Gut Bacteria with Variable Metabolic Priorities for Host Mucosal Glycans MBio. 2015 Nov 10;6(6):e01282-15