Pharmaceutical Microbiology Lecture Notes PDF

Pharmaceutical Microbiology Dr/ Samar Elrefaey R U L E S  Course Content Antimicrobial Agents Sterilization  Antimicrobial Chemotherapeutic Agents lec1 Part 1 The terminology ‘antibiotic’ etymologically signifies anything against life. An antib...

Pharmaceutical Microbiology Dr/ Samar Elrefaey R U L E S  Course Content Antimicrobial Agents Sterilization  Antimicrobial Chemotherapeutic Agents lec1 Part 1 The terminology ‘antibiotic’ etymologically signifies anything against life. An antibiotic was originally defined as a substance, produced by one microorganism, which inhibited the growth of other microorganisms. The advent of synthetic methods has, however, resulted in a modification of this definition and an antibiotic NOW refers to a substance produced by a microorganism, or to a similar substance (produced wholly or partly by chemical synthesis), which in low concentrations inhibits the growth of other microorganisms. The antibiotics and synthetic anti-infective agents are used to treat infections caused by bacteria, fungi and protozoa. Most exert a highly selective toxic action upon their target microbial cells but have little or no toxicity towards mammalian cells. They can be administered at concentrations sufficient to kill or inhibit the growth of infecting organisms without damaging mammalian cells. By comparison, the disinfectants, antiseptics and preservatives are too toxic for systemic treatment of infections.  ‘Magic Bullet’ Cause greater harm Toxicity Selective to microorganisms than to host Antibiotics may either kill (bactericidal) or inhibit (bacteriostatic) the growth of bacteria. According to spectrum of activity, antibiotics are divided into: Broad spectrum Narrow spectrum  The most commonly used antibiotics exert their effect by one of the following methods: 3- 4- 5- 1- 2- Interference Interference Interference Inhibition of Disruption with nucleic with with the cell wall of cell protein acid metabolic synthesis membranes synthesis synthesis pathway Antibiotics belonging to groups 1 and 3 are better able to discriminate between prokaryotic and eukaryotic cells, More selective toxicity and higher therapeutic index.  Classification of antibiotics according to their mode of action: 1. Inhibitors of cell wall synthesis. 2. Inhibitors of protein synthesis. 3. Inhibitors of nucleic acid synthesis. 4. Disruption of cell membrane synthesis. 5. Antifolate metabolism. Antibiotics Mode Of Action Group 1: Inhibitors of cell wall synthesis The main groups which work in this way are the β-lactam antibiotics, so-called because they contain a β-lactam ring in their structure. Included among this group are the penicillins and the cephalosporins. The basal structure component of cell wall is a complex polymer called peptidoglycan consisting of polysaccharides and a highly cross linked polypeptides. About 50% of the weight of the wall of Gram- positive bacteria is peptidoglycan, smaller amounts occur in mycobacterial walls (30%) and Gram-negative bacterial cell walls (10–20%). The polysaccharides regularly contain the amino sugar N- acetylglucosamine (NAG) and N-acetylymuramic acid (NAM) linked alternately in a chain. The final rigidity of the cell wall is imparted by cross linking of peptide chains (through pentaglycine bonds) as a result of transpeptidation reactions carried out by several enzymes. The steps of peptidoglycan biosynthesis involving biosynthesis of soluble intermediate precursors for the peptidoglycan in cytoplasm, assembly of disacharides with short polypeptide chain on a lipid carrier molecules in the cell membrane, Bacterial cell wall structure In the presence of a variety of antibiotics, growing bacteria forms defective cell wall and as a result they undergo lysis and die (bactericidal action). However, these antibiotics would have no effect on the growth of organisms lacking cell wall e.g. L- forms and mycoplasma.  1. ΒETA-LACTAM ANTIBIOTIC http://upload.wikimedia.org/wikipedia/commons/thumb/d/d8/Beta-lactam_antibiotics_example_1.svg/230px-Beta-lactam_antibi Core structure of Penicillins http://upload.wikimedia.org/wikipedia/commons/thumb/d/d8/Beta-lactam_antibiotics_example_1.svg/230px-Beta-lactam_a Core structure of cephalosporins β-Lactam antibiotics are antibiotic agents that contains a β-lactam ring in their molecular structures. This includes Penicillin derivatives (penams), Cephalosporins (cephems), Monobactams, and Carbapenems. Medical use At first, β-lactam antibiotics were mainly active only against Gram-positive bacteria, yet the recent development of broad-spectrum β-lactam antibiotics active against various Gram-negative organisms has increased their usefulness. This irreversible Penicillin and most inhibition other β-of the PBPsantibiotics lactam prevents the act final by cross linking inhibiting penicillin-binding (transpeptidation) proteins, which normallyof the nascent catalyze peptidoglycan cross-linking layer, of disrupting cellwalls. bacterial cell wall synthesis. As a result, the newly synthesized bacterial cell wall is no longer cross-linked and cannot maintain its strength. The transpeptidase enzymes are usually referred to as penicillin- binding proteins (PBPs) A.Penicillins: All penicillins are derived from 6-Aminopenicillanic acid. The various penicillins differ in their side chain structure. Classification of penicillins: Penicillins are divided into Natural and Semisynthetic 1. Natural penicillins Those produced by fermentation of moulds such as Penicillium notatum and P. chrysogenum. The most important examples are: Benzylpenicillin (penicillin G) Phenoxymethylpenicillin (penicillin V). Prototype is penicillin G ❑ pH sensitive. Therefore not given orally. ❑ Effective against Gram-positive cells. ❑ Susceptible to penicillinase. ❑ Spectrum of activity is: ✓ G+ cocci: Pneumococci , Staphylococci, Streptococci. ✓ G- cocci: Meningococci and gonococci. ✓ G+ bacilli: Bacillus perfringens, bacillus diphtheriae. ✓ Spirochetes: Treponema pallidum ✓ Leptospira and Actinomyces. Unsatisfactory features of natural penicillins: ▪ Narrow spectrum. ▪ Easily inactivated. ▪ Has to be given parentally. ▪ Rapidly excreted in urine and therefore the blood level falls quickly unless a dose is injected every 3 hours. Then either: ✓ Given with probencid which blocks renal tubular excretion of penicillins. ✓ Using slowly absorbed compounds such as procaine penicillin. ▪ Since the regular use of penicillin, the number of resistant strains has enormously increased. Acid-stable Penicillins- penicillin V The oral form of penicillins, Narrow antimicrobial spectrum. 2. Semisynthetic penicillins: a.Penicillinase-resistant penicillins: methicillin, oxacillin, cloxacillin and dicloxacillin. b. Broad-spectrum penicillins: ampicillin and amoxicillin ❑ They are similar but weaker to penicillin G in the activity against Gram-positive. ❑ They are similar to chloramphenicol in the activity against Gram-negative organisms. ❑ They are acid-resistant but are not penicillinase- resistant. ❑ Pseudomonas aeruginosa fail to respond to these drugs. c. Antipseudomonal penicilins: carbenicillin, piperacillin and ticarcillin ❑ Extend the ampicillin spectrum of activity to P. aeruginosa and enterobacter species. But their activity to G+ cocci is less than that of ampicillin. ❑ They are not acid-resistant and not penicillinase-resistant. ❑ Generally used in combination with an aminoglycoside for pseudomonal infections. B. Cephalosporins & Cephamycins The cephalosporins are derivatives of 7- amino- cephalosporanic acid. They are relatively stable in dilute acid and are highly resistant to penicillinase. Spectrum of activity and clinical uses of cephalosporins ▪ All cephalosporins are active against most G+ cocci, including penicillinase-producing staphylococci and many strains of G- bacilli, but relatively ineffective against enterococci. ▪ Divided into 5 major groups called “Generations'" based on parallel their chronological development their antimicrobial spectrum. a. First Generation Cephalosporines: cephalothin, cefazolin, and cephalexin ❖ They have a stronger antimicrobial action on G+ bacteria than that of the other generations, but their action on G- bacteria is relatively poor. ❖ They are NOT effective against pseudomonas. ❖ Comparatively, they are stable for beta- lactamase (penicillinase). ❖ They are chiefly used in treating infection of the penicillinase-productive aurococcus (S.aureus) and surgical prophylaxisction. ❖ Non of the first generation penetrate CNS or reach CSF. b. Second Generation Cephalosporins: cefamandole, cefoxitin, cefaclor, cefuroxime, cefprozil. ▪ Action of this generation on G+ bacteria is the same or a little bit less than that of the first generation. ▪ Their antimicrobial action on G- bacteria is obviously increased. ▪ Some of them are effective against anaerobes such as B. fragilis ▪ Ineffective against P. aeruginosa. ▪ Cefuroxime is the only second-generation drug crosses the BBB. ▪ They are stable to many kinds of beta-lactamases and have less nephrotoxicity than the first generation. ▪ Dosage must be reduced in renal failure. c. Third Generation Cephalosporins: cefotaxime, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefixime ✓ The highest activities against G- bacteria, the lowest activities against G+ bacteria. ✓ The highest resistance to β-lactamase. ✓ The best penetration into the CSF; almost no nephrotoxicity. ✓ Ceftizoxime has good activity against B. fragilis. ✓ Ceftazidime is the best anti-pseudomonal cephalosporin. d. Fourth Generation Cephalosporin: Cefipime ▪ Cefipime has activity comparable to that of ceftazidime against P. aeruginosa. ▪ The activity against streptococci and methicillin- susceptible staphylococci is greater than that of ceftazidime and comparable to that of the other third generation compounds. e. Next generation cephalosporin: Ceftaroline ▪ Broad spectrum; active against the common Gram-negative bacteria, some Gram-positive activity (drug resistant S. pneumoniae). ▪ Notable for activity against MRSA, unlike any other beta-lactam antibiotic. BREAK  Antimicrobial Chemotherapeutic Agents lec1 Part 2  C. The other beta-lactam antibiotics: i. Monobactams :Aztreonam Monobactams are β-lactam compounds wherein the β-lactam ring is alone and not fused to another ring. ▪ Aztreonam is highly resistant to beta-lactamases as not hydrolyzed by ß-lactamases. ▪ It is highly active against aerobic G- bacteria, including P. aeruginosa and penicillinase producing strains of H. influenzae. ▪ But it shows poor activity against Aztreonam. G+ cocci and anaerobic bacteria. The square is the β-lactam. There is a second thiazole ▪ The antimicrobial spectrum of ring, but it is not fused to the aztreonam is similar to that of β-lactam ring.) aminoglycosides.  ii. Carbapenems 1. Imipenem is stable to most beta-lactamases but is readily hydrolysed by kidney dehydropeptidase to a nephrotoxic metabolite and is administered with a dehydropeptidase inhibitor, cilastatin. The mixture is called tienam. The antimicrobial spectrum of imipenem is the broadest one of all the beta-lactam antibiotics. It is active against G+, G- cocci (EXCEPT methicillin resistant staphylococci), enterobacteriaceae, P. aeruginosa, and anaerobic bacteria. Imipenem could NOT be used in CNS infections. carbapenem 2. Meropenem is more stable than imipenem to this enzyme and may thus be administered without cilastatin. 3. Ertapenem has properties similar to those of meropenem but affords the additional advantage of once-daily dosing. iii. Carbacephems Loracarbef is highly active against Gram- positive bacteria. iv. Nocardicins The nocardicins (A to G) have been isolated from a strain of Nocardia and comprise a novel group of beta-lactam antibiotics. Nocardicin A is the most active member, and possesses significant activity against Gram- negative but NOT Gram-positive bacteria. Other inhibitors of cell wall synthesis 1. GLYCOPEPTIDES A. Glycopeptide: Vancomycin Vancomycin is a glycopeptide antibiotic produced by Streptococcus orientalis. It is poorly absorbed from the intestine Orally only for the treatment of antibiotic associated pseudomembranous colitis caused by C. difficile. Mechanism of action: Binds to precursor units of bacterial cell walls, inhibiting cell wall synthesis by: 1. Block the transglycosylation reaction (insertion of the disaccharide pentapeptide into the cell wall at a growing point by a transglycosylase) 2. Binds to the building blocks (NAG and NAM) of the peptidoglycan and prevents the transpeptidase from acting. B. Lipoglycopeptide ▪ Dalbavancin Bactericidal for MRSA ▪ Oritavancin 2nd gen. glycopeptide. It's once per day dosing. Activity similar to vancomycin and better for Staphyloccus and Enterococcus. ▪ Telavancin: bactericidal for all Gm- positive ▪ Teicoplanin It also binds tightly to the D-alanyl-D-alanine region of the peptidoglycan precursor. 2. BACITRACIN Bacitracin is a polypeptide obtained from a strain of Bacillus subtilis. It is stable and poorly absorbed from the intestinal tract. Its only use is for topical application to skin, wounds or mucous membranes. Mechanism of action: Interfere with the lipid carrier molecules. Bacitracin is mainly bactericidal for Gram-positive bacteria, including penicillin-resistant staphylococci. In combination with polymyxin B or neomycin, bacitracin is useful for the suppression of mixed bacterial flora in surface lesions. Bacitracin is toxic for the kidney. For this reason, it has no place in systemic therapy. 3. CYCLOSERINE Because of its structural similarity to D- alanine, it interferes with the production of D-alanine. This occurs inside the cytoplasm and involves a racemase enzyme which converts L-alanine to D-alanine and a ligase which couples two D-alanines together 4.FOSFOMYCIN Fosfomycin inhibits the synthesis of NAM. It is active against Gram +ve and –ve spectrum.  BETA-LACTAMASE INHIBITORS -Inactivate bacterial beta-lactamases and are used to enhance the antibacterial actions of beta-lactam antibiotics. -Only have weak antibacterial action. Mechanism of action: The inhibitors has a strong affinity for β-lactamases and are hydrolysed by the beta-lactamases in the same manner as susceptible beta-lactam antibiotics. It is thought that the hydrolysed inhibitors can interact with a second enzyme residue in the active site of the beta-lactamase, forming a covalently cross-linked, irreversibly inhibited complex. Clavulanic acid isolated from Streptomyces clavuligerus, has poor antibacterial activity but is a potent inhibitor of β-lactamase. Tazobactam and sulbactam synthetically produced. Clavulanic acid, sulbactam and tazobactam have been developed for use in combination with susceptible beta-lactams (amoxycillin, ampicillin and piperacillin, respectively). Inhibitors are active against all penicillinase but never on cephalosporinase Mycolic acid and arabinogalactan biosynthesis in mycobacteria and fungi The lipid-rich nature of the mycobacterial wall serves as a penetration barrier to many antibiotics. 1. Isoniazid Isoniazid interferes with mycolic acid synthesis by inhibiting an enoyl reductase which forms part of the fatty acid synthase system. 2. Ethambutol Ethambutol is thought to block assembly of the arabinogalactan polysaccharide by inhibition of an arabinotransferase enzyme. 3.Echinocandins Caspofungin interferes with the synthesis of the b-1,3-D-glucan polymer in the fungal cell wall. Without the glucan polymer, the integrity of the fungal cell wall is compromised, yeast cells lose their rigidity and become like protoplasts.  INHIBITORS OF PROTEIN SYNTHESIS Bacterial protein synthesis differs in several respects from that occurring in animal cells. In particular the ribosomes have different characteristics. Those from bacteria have a sedimentation coefficient of 70(70S) and give rise to 30S and 50S subunits. The mammalian ribosomes are 80S with subunits of 40S and 60S. The therapeutic success of antibiotics which interferes with protein synthesis depends upon a strong selectivity toward the bacterial system.  Inhibitors of protein synthesis: (c) Chloramphenicol inhibits peptidyl- transferase and prevents formation of E P A new peptide bonds. 50s subunit (d) Macrolides such as erythromycin (b) bind to the 50S Tetracyclines subunit, tRNA preventing distort the elongation of the shape of the 30S growing peptide 5’ subunit, chain 3’ preventing the attachment of mRNA the appropriate 30s subunit aminoacyl tRNA. (a) Binding to the 30S subunit of the bacterial ribosome, aminoglycosides block the attachment of the 50S subunit. This prevents completion of the initiation complex, thus protein synthesis is inhibited. A. Antibiotics binding to 30s ribosomal subunits I.AMINOGLYCOSIDES Steps of bacterial protein synthesis. Streptomycin inhibits protein chain initiation Puromycin, chloramphenicol, cycloheximide, and tetracycline inhibit protein chain elongation Aminoglycosides that are derived from bacteria of the Streptomyces genus are named with the suffix -mycin, whereas those that, gentamicin, are derived from Micromonospora are named with the suffix -micin. Others later developed amikacin, netilmicin, tobramycin Mechanism of action: 1. Prevent the formation of an initiation complex of peptide formation by preventing the binding of initiator tRNA to the P-site of the initiation complex. 2. Cause misreading of the messenger RNA message, leading to the production of nonsense peptides. Streptomycin binding to the 30S subunit distorts the shape of the A site on the ribosome and interferes with the positioning of the aminoacyl-tRNA molecules during peptide chain elongation. 3. Increase membrane leakage: a. As streptomycin exerts a potent lethal action, by formation of toxic, non- functional proteins through misreading of the codons on mRNA is a more likely mechanism of action b. Since other antibiotics that inhibit the synthesis of proteins (such as tetracycline) are not bactericidal. Humans do synthesize proteins, but at a much slower rate than bacteria, so drug can act on bacteria before it does damage to man. General characters: ❖ Bactericidal to sensitive organisms. ❖ Not absorbed by mouth. ❖ May be nephrotoxic. ❖ All are potentially neurotoxic. ❖ Occupy extra cellular space, little penetration of CSF Spectrum of activity: a. Broad Gram negative coverage including Pseudomonas. i- Almost always used in combination with another antibiotic such a beta-lactam to extend coverage, provide synergy. ii- Penicillin and aminoglycoside antibiotics must NEVER be physically mixed, because both are chemically inactivated to a significant degree on mixing. b. Streptococci are relatively impermeable to aminoglycosides. c. Negligible anaerobic coverage, because transport through the cell membrane is an energy requiring process that is oxygen- dependent. d. Exhibit a post-antibiotic effect in which there is no or very little drug level detectable in blood, but there still seems to be inhibition of bacterial re-growth. This is due to strong, irreversible binding to the ribosome, and remains intracellular long after plasma levels drop. Streptomycin 2nd line for tuberculosis in combination with other agents. Gentamicin (Garamycin) It is more active than other members against a wide range of bacteria. Effective for both Gm+ve and Gm-ve. Almost always used in combination with another antibiotic (beta-lactam) as carbenicillin to delay development of resistance. Tobramycin (Nebcin) Similar coverage overall to gentamicin, except better Ps. coverage. There is some cross-resistance between tobramycin and gentamicin. Netilmicin (Netromycin) Similar to gentamicin and tobramycin, but may be more active against resistant strains Amikacin (Amikin) It is a semisynthetic derivative of kanamycin. Used for resistant bacteria because it is resistant to several of the enzymes that inactivates gentamicin and tobramycin. Spectinomycin (Trobicin) Aminocyclitrol antibiotic structurally related to aminoglycosides (lacks amino sugars and glycosidic bonds). Active in vitro against Gram + and Gram -. Used clinically as alternative treatment for drug- resistant gonorrhea or gonorrhea in penicillin-allergic patients. Kanamycin (Kantrex), neomycin and paromycin ▪ They are similar with cross-resistance. ▪ Kanamycin is a second line drug in treatment of tubeculosis. ▪ Paromomycin is used in amebiasis. ▪ Oral doses of both kanamycin and neomycins are used for reduction of intestinal flora before large bowel operation often in combination with erythromycin. ▪ Otherwise, these drugs are mainly limited to topical application on infected surfaces because NEITHER of them is used systemically because of oto- and neurotoxicity. 2. TETRACYCLINE GROUP Tetracyclines http://upload.wikimedia.org/wikipedia/commons/thumb/8/84/Tetracyclines.png/250px-Tetracyclines.p The 4 rings of the basic tetracycline structure Tetracyclines are produced by actinomyces, which have broad-antibacterial spectrum. The tetracyclines are a group of drugs with four- ringed structure that differ in physical and pharmacologic characters but have virtually identical antimicrobial properties and give complete cross-resistance. Mechanism of action Tetracyclines inhibit protein synthesis occurring in the smaller subunit either 40S or 30S. However, tetracyclines enter prokaryotic cells (actively transported) and concentrated into its cytoplasm with the aid of a permease, achieving a 50-fold concentration inside the cells. Tetracyclines can also inhibit protein synthesis in the host, but are less likely to reach the concentration required because eukaryotic cells do not have a tetracycline uptake mechanism (small amounts enter by diffusion alone). Binding to 30S subunit inhibits aminoacyl tRNA binding to the A-site and this prevents the introduction of a new amino acid into the polypeptide chain. Tetracyclines are bacteriostatic rather than bactericidal, consequently they should not be used in combination with β-lactams, which require cells to be growing and dividing to exert their lethal action. They are broad-spectrum antibiotics, i.e. they have a wide range of activity against Gm-positive and Gm-negative bacteria. Ps. aeruginosa is less sensitive, but is generally susceptible to tetracycline concentrations obtainable in the bladder. Therapeutic uses ▪ Urinary tract and the intestines infections. ▪ Moderately severe acne to suppress both skin bacteria and their lipases,. ▪ Doxycycline is also used as a prophylactic treatment for infection by Bacillus anthracis and is effective against Yersinia pestis. It is also used for malaria treatment and prophylaxis, as well as treating elephantiasis. ▪ The Drug Of Choice for infections caused by chlamydia, rickettsia, brucellosis, and spirochetal infections (borreliosis, syphilis, and Lyme disease). They may have a role in reducing the duration and severity of cholera Classification of tetracyclines: According to source: ▪ Naturally occurring : Tetracycline, chlortetracycline, oxytetracycline and demeclocycline ▪ Semi-synthetic ; Doxycycline, methacycline and minocycline According to duration of action: ▪ Short-acting (Half-life is 6-8 hrs): Tetracycline, chlortetracycline and oxytetracycline ▪ Intermediate-acting (Half-life is ~12 hrs): Demeclocycline and methacycline ▪ Long-acting (Half-life is 16 hrs or more): Doxycycline and minocycline GLYCYLCYCLINES: Tigecycline ▪ The glycylcyclines are novel tetracyclines substituted at the C-9 position with a dimethylglycylamido side-chain. ▪ They possess activity against bacteria that express resistance to the older tetracyclines by an efflux mechanism. ▪ Used in treatment of complicated skin and skin structure infections caused by susceptible organisms, including MRSA and vancomycin-sensitive Enterococcus faecalis; treatment of complicated intra-abdominal infections. Thanks