microbiology, lecture 10

8/7/2019 Microbiology, Lecture 10

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Today's lecture will be about Antimicrobial agents in general and

next time we will talk about resistance to antimicrobial agents .So

let's begin with some definitions;

t is any drug used for the treatment of anyA chemotherapeutic agen

type of disease.

The chemotherapeutic agent that's used in treatment of microbial

infection is calledAntimicrobial Agent;it either kills the pathogen

or inhibit its growth. Some antimicrobial agent are called

Antibiotics.

organism that'sis a natural substance produced by anAntibiotic

killed or inhibited by another organism.

..As you know organism are killed in the environment in presenceof other microbes ; so in order to this organism to establish them in

the environment ,sometimes they produce substances to kill other

species ,they later discovered these substances and called them

"Antibiotics"; so antibiotic is one example of an antimicrobial agent,

which is an example of chemotherapeutic agent.

Antimicrobial agent can be classified according to what type of

microbe they are effecting:

y antibacterialagent is this to treat bacterial diseases ,y those used to treat fungal diseases, antifungal agents;y those used to treat protozoal diseases, antiprotozoal agents;y those used to treat viral diseases, antiviral agents.

After discovering antibiotics they started using them against

infection but they later found that some bacteria are mutated and

become resistant to antibiotics, so they started modifying antibiotics

and come up with semisynthetic antibiotics ;also one reason for

modifying antibiotic is to give them better properties , for example

the resistance against acidity of the stomach .

One of the first antibiotic to be discovered was Penicillin ;AlexanderFleming discovered that in presence of Penicillin in a media very few

bacteria grow in the media adjacent to it; this is an example of such

environment ,this is Staphylococcus aureus ,one of the bacteria that

grows on the skin and related to some kinds of infections, you can see

that bacteria grow away from the fungus and near it ,it grows very

poorly and they discovered that the fungus is producing penicillin

which is diffusing away from the fungus inhibiting bacterial form.


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So, what are the characteristics of an ideal antimicrobial agent?

y First, a good antimicrobial agent should kill or inhibit thepathogen,

y secondly it should inhibit growth without affecting the host cellwe refer at these two properties as Selective toxicity ;so they areselectively affecting the microbe but not affecting the host tissue,

y A good antimicrobial agent is good when it doesn't lead toallergy , we know some people are allergic to penicillin;

y also a good antimicrobial agent should be stable when storedand the drug need to stay in the affected tissue for a good

period of time and not exiting quickly , so you will not have to

give him multiple doses.

y And finally the agent of the antimicrobial agent needs to bevery effective and achieve the killing and inhibition of growth

without having organism becoming resistant to it.

So these are the characteristics of an ideal antimicrobial agent. Not all

agents have these characteristics ,some of them are toxics ,some are

secreted quickly and so on. Some have side effect s ,and we will talk

about these sides effects.

The history of antimicrobial agent, I don't expect you to know the slide,

it is just to give you an idea about the history ..

the first antimicrobial agent was discovered between

1930-1940 ,discovered the penicillin as I told you from Fungi .

1950 -1960,discovered other antibiotics.

1970-1990s No novel classes were discovered. All the new

antimicrobials discovered were derivatives of previous groups.

Later 2000 - 2005Three new classes were discovered.

So if you want to count how many classes and derivatives of these

classes that exist in medicine you will find that there are almost 50

derivatives of penicillin, 70 cephalosporin.

SELECTIVE TOXICITYis The Central Concept of antimicrobial action,this means that the growth of the infected organism is selectively

inhibited ,or the organism is killed ,without damaging the host tissue.

Looking at the antimicrobial agent function ,these agents can be

classified into Bactericidal or Bacteriostatic .

A bacteriostatic agent : is an agent that inhibit the multiplication of

bacteria or any other organism without killing it ;so we have no more

increase in bacterial number or fungal number or protozoal number


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;and here you clean the infection through the action of the immune

system. So if we have patient with very good immunity you can give

him a bacteriostatic agent and that should be enough. The

bacteriostatic agent will stop the growth and the immune system will

clean all the damaged bacteria. Alternatively, soma antimic robial

agent are bactericidal , and these, actually, kill the organisms .So bactericidal agents are good for people with weakened immune

system; people with HIV, cancer, leukemia, you have to give them an

bactericidal agent because they cannot clear the infection without it.

Chemically speaking antimicrobial agent have different types of

chemistry and different modes of action and I dont want you to

memorize these names, referring to slide 12.

So the way antimicrobial agents achieve selective toxicity is by

targeting a specific metabolic step or a specific application step of an

orgaism that's not present in human cell or animal cell . For example

the cell wall is a very good and very important target for fightingmicrobial infection. Bacteria, as you know , have a cell wall whereas

animal cell lack cell wall ;so if you develop a target for cell wall that's

a very good target .

Another example of a target is "cell membrane", one group of

Polymyxins , that selectively can target the cell membrane of bacter ia

without affecting the membrane of animal cells.

Also we can target DNA replication something , some drugs called

Quinolones , and affect the enzymes for DNA Gyrase only present in

bacteria but not present in animal cells. Again , we can target the step

of infection ,so DNA directed RNA Polymerase ,in short R.polymerasecan be affected by Rifampin , but the animal or euckaryotic will not be

affected by this target. And then we have another group of other

enzymes that can target variant steps in protein synthesis, such as

Macrolides , Chloramphenicol ,Clindamycin, Tetracycline,

Spectinomycin and Aminoglycosides ; and finally we can also target

variant metabolic pathways ,for example ,the pathway that we lead to

folic acidcan be metabolized by two antibiotics Trimethoprim and

Sulfonamides .

So The 5 most common targets of antimicrobial agents are:

I. Inhibition of cell wall synthesis

II. Damage to cell membranes

III.Inhibition of nucleic acid synthesis

(either duplicating DNA or RNA )

IV.Inhibition of protein synthesis

V. Inhibition of enzyme activity of metabolic pathways.


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So here are the Major Categories of Antibacterial Agents ,and I expect

you to memorize themso

y Penicillins and Cephalosporins both interfere with cell wallsynthesis ,and both them are bactericidial , meaning they

actually kill the bacteria.y Tetracyclines is bacteriostatic , meaning that they only inhibit

the bacterial growth and they inhibit protein synthesis.

y Aminoglycosides again inhibit protein synthesis but they arebacteriocidial ;

y Macrolides are bacteriostatic at lower doses and bactericidal athigher doses and also inhibit protein Synthesis.

y Finally Quinolones which are bactericidal and inhibit DNAsynthesis.

So those are the most important examples to remember regarding

the mode of function of antimicrobial agents.

So let's assume that we have a tube in which a culture medium in

which bacteria is growing ;as I mentioned to you ,bacteria growing in

liquid medium will lead to the formation of turbidity or cloudiness in

the medium. So this is the turbidity of the tube over time for a variety

of antibiotic so the turbidity will rise until it reaches a threshold in

which we have no more bacterial growth. So this is basically

temporal phase .Let's assume at this time here, indicated by the

arrow, is added one type of antimicrobial agent ,you will find that

variant antibiotics lead to variant effect without bacterial growth

;you will find that:

y Penicillin ,added here(pointing to the slide ) bacteria continueits growth for a short of time but eventually immediately fall.

y Streptomycin ,Chloramphenicol ;streptomycin is an example ofaminoglycoside ,you add it at this time point and immediately

you have no more bacterial growth .

y Sulfonomide you add it here ,bacteria continue to grow for agood amount of time and eventually stop multiply.

So you can see that different antibiotics or different antimicrobial

agent have different mode of action and I described to you the

variant mode of action of these and if you acquire these mode of

actions you will be able to interpret why this types of pattern

happen.


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So looking at bacterial growth in general , if we look at viability

meaning how many bacteria are still alive in the tube ,you will find

that Streptomycin and Pencillin kill all bacterial cell ,so no bacteria

remained alive in the tube , whereas Chloramphenicol didn't kill any

cell only inhibited the growth ,so if Chloramphenicol was removed, in

some way, Bacteria will continue to grow again .Sulfanomide alsodidn't kill the bacteria only inhibited further multiplication.

So Streptomycin and Pencillin are bacteriocidial and

Chloramphenicol and Sulfanomide are bacteriostatic.

So will begin with first antimicrobial agent which is -lactams.

-lactams antibiotic:

So a -lactams antibiotic is an antimicrobial agent that's contained in

the structure of all these (listed in slide 18) ,for this we call these -

lactams. An example of -lactams is penicillin and Cephalosporin .

The very firstPencillin were derived from Fungi and the name of the

natural penicillin is Penicillin G and Penicillin V.(their chemical name

,which is written in the slide within brackets is not for

memorization).

So these penicillin have limited spectrum ,meaning that they only

inhibit the growth, they only kill ,Gram positive bacteria they don't

affect Gram negative bacteria and they are also -lactamase sensitive.

So I told you that many bacteria and many organisms are now

developing resistance to antimicrobial agents ,one way the

organisms are resisting the -lactamas antibioticsthe penicillin andCephalosporin is by producing an enzyme that cut the -lactam base

,so by cutting the ring the antibiotic is no more functional. So these

drugs were -lactamase sensitive ,so if bacteria produces an enzyme

the drug will be inactivated and bacteria will continue growing.

Scientists took the early antibiotics and start modifying the groups

around the -lactam ring without affecting the -lactam function and

they came up with Ampicillin and Amoxicillin . They are Broader

spectrum they can kill Gram positive and Gram negative bacteria

.They also modified the chemical groups and came up with Methicillin

and Oxacillin in addition to being broad spectrum they are -

lactamase resistant ,they are able to survive in presence of -

lactamases ,they are not easily inactivated by -lactamase ;

Methicillin are alsoAcid labile ,meaning you can't take it through

capsule format ,you have through vein ,because it will destroy the

antibiotic. Whereas Oxacillin is acid stable ,meaning we can get this

drug orally. Finally , we have Carbenicillin ,this is an Extended


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spectrum, functioning against Gram positive and Gram negative

bacteria and against a very important pathogen for Pseudomonas. So

Pseudomonas is a resistant to many drugs and only a few

antimicrobial agents can kill it.

So the mode of action of Penicillin or -lactam in general ,applied topenicillin and cephalosporin.

When we were talking about cell wall structure, we said that we have

chains of repeating dysugars N- Acetylglucosamine linked to N-

acetylmuramic acid and from N-acetylmuramic acid there was

another amino acid chain and attached to some of these there was a

pentaglysine bridge attaching between adjacent peptide chains

forming very rigid cross bridges. So the attaching of pentaglycine

with the adjacent chain is done by an enzyme called Transpeptidase

,which is called Penicillin binding protein ;so penicillin can bindthis enzyme and inhibit its function; so in presence of penicillin

bacteria will not be able to create new b ridges in the cell wall ,

existing bridges will not be affected only new ones . So if you have

bacteria and the bacteria is not multiplying ,is not growing ,and you

add penicillin to it ,nothing will happen; only when bacteria is

multiplying penicillin will have effect .So in order to multiply, the

cross bridges in the cell wall will be broken up ,the cell wall

dissociate and reattached properly. so this is how bacteria grow ,so

you have to break some bonds and then reattach them .If a bacteria

is growing and it meets penicillin the cross bridges will be breakingbut not be forming and because the cell wall is a rigid structure

protecting the cell from high external osmotic pressure ,eventually

the bacteria will burst, because the cell wall become extrem ely weak

.so that's the mode of action of penicillin.

SO INSHORT: PENICIILIN PREVENT THE FORMATION OF NEW

PEPTIDE CORSS DRIDGES IN THE CELL WALL ,WEAKINING OF THE

CELL WALL LEADING TO THE LYSIS OF BACTERIAL CELL DUE TO

HIGHT EXTERNAL OSMOTIC PRESSURE.

Cephalosporin :

These are again -lactam drugs ,function similarly to penicillin, they

affect Transpeptidase , but in contrast to penicillin they are broader

in spectrum ,meaning that they are active against more and more

bacteria, they are natural resistant to some -lactamases ;the

disadvantages of cephalosporin is that they are less potent meaning


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that you have to get more and more cephalosporin to get the effect as

penicillin in addition to being more expensive.

So cephalosporin have longer generation depending on what

chemical modifications they have ;so early cephalosporin were

derived from bacteria ,Gram positive bacteria, then they started

simply modifying the antimicrobial agents and came up with second-generation cephalosporin which have expanded spectrum against

Gram positive and Gram negative bacteria and Higher resistance to

-lactamase .The third-generation ,again has Wide spectrum and a

greater activity against Gram negative bacteria ,including

Pseudomonas; finally you have the fourth generation which also

have very wide spectrum .

So these terms, wide spectrum expanded spectrum increased

spectrum.. ,all refer to having wide range of action.

Slide 24 shows some examples of the 4 generations of cephalosporin

,you don't have to memorize these names.And here is summary of some Beta-Lactam antibiotics ,slide 25,and

the various properties ,they are not for memorizing.

All drugs that target synthesis of cell wall require growth of the

organism and of them are considered to be Bactericidal; so if you put

any of these drugs in a test tube , all them will give you a similar

pattern of penicillin, all of them will inhibit growth and kill bacteria .

After cell wall we have cell membrane:

We have a group of drugs called Polymyxin ,these Polymyxin simplyinsert themselves in the cell membrane creating a channel through

which the cytoplasm can leave to the outside and the bacterial cell

will die.

So Polymyxinare bacteriocidial agent.

I also have two example ofANTIFUNGAL AGENTS : we have

amphotericin it induct itself in fungal cell membrane and leads to

the leakage of the cytoplasm ; Imidizoles ,also interfere with

ergosterol biosynthesis ; so fungal cell wall don't have cholesterol

like human cell or animal cell, have something called ergosterol ,so

imidizole prevent the synthesis ofergosterol and compromising the

cell membrane of fungi leading to the death of fungi.

Protein Synthesis Inhibitors these are important group of drugs .

In order to break a protein first we have to create a copy of the gene

in the form of mRNA ,mRNA will bind to 30S subunit of ribosome


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and start transcripting the mRNA into protein ;so this process involve

many steps and variant antimicrobial agent can target the variant

steps of protein synthesis.

All Protein Synthesis Inhibitorsare bacteriostatic except

Aminoglycosides ,these are bactericidal .

Aminoglycosides ,these drugs bind irreversibly to 30S subunit of the

ribosome and prevent the synthesis of protein ; they bind

irreversibly so all ribosomes in the bacterial cell will be inhibited and

the cell will die ,because can't make protein so it become not

functional.

These drugs are broad spectrum ,affect Gram positive and Gram

negative and they were found to be Synergistic with Penicillins ;so if

you give a patient an aminoglycoside and penicillin at the same time

you will end up with high risk of killing ,higher than aminoglycosidealone or higher than penicillin alone.

The remaining inhibitor of protein synthesis most of them bind to the

50S subunit of the ribosome with exception Tetracycline which binds

30S subunit and Spectinomycin binds 30S subunit.

Remember 30S :

y Tetracycline,y Spectinomycin ,y Aminoglycoside

All other target the 50S subunit .

Tetracycline are natural antibiotic ,derived from yeast organisms

;later on they started modifying the chemical groups of this group in

base on the structure of Tetracycline to give them other properties.

These are broad spectrum and these are the best choice with

infection with Rickettsia, Chlamydia ;Tetracycline are actively

transported inside bacterial cell ;most bacteria have Tetracycline

outside and they are transported inside.

Chlorophenicol is another example of a protein synthesis inhibitor

,this drug reversibly bind to 50S subunit of the ribosome and it's

bacteriostatic and broad spectrum .

So you only give Chlorophenicol in extreme case when you have no

other choice ,because it toxic to human and might be cause Aplastic

anemia . This is one drug which have a lot of side effects.

Lincomycin and the derivative Clindamycin ,


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These are inhibitors and ,these are effective against anaerobic

bacteria e.g. Bacteriodes fragilis .

Macrolides basically the Erythromycin:

Primarily effective against Gram positive bacteria and these are the

best choice with infection with Mycoplasma pneumoniae ,which isthe cause of Primary Acidic Pneumonia, and Legionaires Disease

that is a very serious side of pneumonia.

Nucleic Acid Synthesis

Rifamycins e.g. Rifampin ,these inhibit RNA synthesis by inhibiting

the bacterial RNA polymerase and not affecting the eukaryotic RNA

polymerase .

Quinolones ,I mentioned it earlier in the lecture ,these drugs inhibit

the DNA Gyrase and you know DNA in bacterial cell is super coiledhighly compacted and a DNA polymerase ,RNA polymerase can't

start copying the DNA unless the DNA is relaxed ;so a DNA Gyrase

basically relaxes the super coiled DNA .So by putting Quinolones ,you

inhibit the DNA Gyrase and the cell can't perform any of its function

and cannot replicate.

This drug is bactericidal, Broad spectrum ,meaning they are active

against Gram positive and Gram negative and also effective against

pseudomonas aeruginosa, Methicillin-resistant Staphylococcus

aureus.

Almost done,few more slides We have other drugs that can target other metabolic pathways within

bacterial cells .

One of these pathways is Tetrahydrofolic acid synthesis pathway

PABA + Pteridine in presence ofSynthetase enzyme will be

converted into this compound , Dihydropteric acid, and then by

Dihydofolate reductase will be converted into Tetrahydrofolic acid .

Some drugs were discovered to inhibit this enzyme( the Synthetase

and reductase) ;and by doing so the cell cannot produce

Tetrahydrofolic acid which is very important for the cell survival; so

Thymidine, Purines and Methionine all depend on tetrahydrofolic

acid .

So if we give Sulfonamides alone or Trimethoprim alone, the cell will

basicly stop dividing ,so the effect will be bacteriostatic but if you


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give these two drugs together as a combination the effect will be

bactericidal ,potentially bactericidal .

We already talked about penicillin ,so Streptomycin is an example of

aminoglycoside ,once you add it they will immediately bind to the

ribosome and the cell is immediately unable to make protein ,so itcannot divide any further,that's why we have low turbidity and low

viability( low growth).

Chloromphenicol is again a bacteriostatic inhibitor can prevent

protein synthesis .

If you look at how many cells are alive and how many cells are dead

,you find that Streptomycin actually killed the bacterial cell because

the cell is no more able to make any protein but Chloromphenicol is

bind reversibly with the 30S subunit of the ribosome the cell is

actually still alive but not multiplying.

Sulfonimide are the most interesting ones ,because you see thatbacteria continue to grow for a time and eventually stop growing

;why is this happening? The cell in presence of sulfanomide can't

make additional tetrahydrofolic acid ,but actually the cell has a small

amount of tetrahydrofolic acid still present in the cytoplasm ,the cell

have small amount of Thymidine, Purines, Methionine present in the

cutoplasm ;so the cell will continue to grow and multiply until all o f

these are exhaustedonce they are exhausted it will can't take more

of them and the cell will stop growing .

Again regarding Sulfonimide are bacteriostatic but can become

bacteriocidial .

The last agent for today is Metronidazole :

You as dentists ,will be using Metronidazole a lot to treat a lot of

infections for anaerobic bacteria ,so Metronidazole enter the

bacterial cell become in the active form ,once reduced it will be able

to destroy and fragment the DNA ;once DNA is fragmented the cell is

dead . Metronidazole is a bactericidal agent.

Done by Sara Ibdiwi

microbiology, lecture 10

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