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; A chemotherapeutic agen t is any drug used for the treatment of any type of disease. The chemotherapeutic agent that's used in treatment of microbial infection is called Antimicrobial Agent ;it either kills the pathogen or inhibit its growth. Some antimicrobial agent are called Antibiotics. Antibiotic is a natural substance produced by an organism that's killed or inhibited by another organism. ..As you know organism are killed in the environment in presence of other microbes ; so in order to this organism to establish them in the environment ,sometimes they produce substances to kill o ther 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 antibacterial agent 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 ;Alexander Fleming 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.

So, what are the characteristics of an ideal antimicrobial agent? y First, a good antimicrobial agent should kill or inhibit the pathogen, y secondly it should inhibit growth without affecting the host cell we refer at these two properties as Selective toxicity ;so they are selectively affecting the microbe but not affecting the host tissue, y A good antimicrobial agent is good when it doesn't lead to allergy , we know some people are allergic to penicillin; y also a good antimicrobial agent should be stable when stored and 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 be very effective and achieve the killing and inhibitio n 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 - 2005 Three 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 TOXICITY is 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

;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 i nfection without it. Chemically speaking antimicrobial agent have different types of chemistry and different modes of action and I don t 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 fighting microbial 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.polymerase can 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 acid can 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.


So here are the Major Categories of Antibacterial Agents ,and I expect you to memorize them so y Penicillins and Cephalosporins both interfere with cell wall synthesis ,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 are bacteriocidial ; y Macrolides are bacteriostatic at lower doses and bactericidal at higher doses and also inhibit protein Synthesis. y Finally Quinolones which are bactericidal and inhibit DNA synthesis. 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 continue its growth for a short of time but eventually immediately fall. y Streptomycin ,Chloramphenicol ;streptomycin is an example of aminoglycoside ,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 a good 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.


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 also didn'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 ß-lactams. ß-lactams antibiotic: So a ß-lactams antibiotic is an antimicrobial agent that's contained in the structure of all these (listed in slide 18) ,for this we call these ß lactams. An example of ß-lactams is penicillin and Cephalosporin . The very first Pencillin 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 ß-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 ß-lactamas antibiotics the penicillin and Cephalosporin is by producing an enzyme that cut the ß-lactam base ,so by cutting the ring the antibiotic is no more functional. So these drugs were ß-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 ß-lactam ring without affecting the ß-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 ßlactamase resistant ,they are able to survive in presence of ßlactamases ,they are not easily inactivated by ß -lactamase ; Methicillin are also Acid 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


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 ß-lactam in general ,applied to penicillin and cephalosporin. When we were talking about cell wall structure, we said that we have chains of repeating dysugars N- Acetylglucosamine linked to Nacetylmuramic 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 bind this 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 breaking but 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 IN SHORT : 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 ß-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 ß-lactamases ;the disadvantages of cephalosporin is that they are less potent meaning


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 secondgeneration cephalosporin which have expanded spectrum against Gram positive and Gram negative bacteria and Higher resistance to ß-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 simply insert themselves in the cell membrane creating a channel through which the cytoplasm can leave to the outside and the bacterial cell will die. So Polymyxin are bacteriocidial agent. I also have two example of ANTIFUNGAL 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 of ergosterol 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


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 Inhibitors are 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 aminoglycoside alone 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 ,

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 is the 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 coiled highly 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 of Synthetase 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


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 it cannot 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 that bacteria 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 exhausted once 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


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