Controlling microbial growth in vivo by using antimicrobial agents. Dr. Nasser M. Kaplan Medical Microbiology JUST, Irbid, Jordan
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1 Controlling microbial growth in vivo by using antimicrobial agents Dr. Nasser M. Kaplan Medical Microbiology JUST, Irbid, Jordan
2 Introduction Definitions Chemotherapeutic agent = any chemical (drug) used to treat any disease (condition). Antimicrobial agent = chemotherapeutic agent used to treat IDs as antibacterial, antiviral, antifungal & antiprotozoal agents. Hx Paul Ehrlich (German chemist): father of chemorx (Arsenic for syphilis). Alexander Fleming (Scottish Bacteriologist): Penicillium notatum penicillin. 11-Nov-15 NM Kaplan 2
3 cccs of IDEAL antimicrobial agent 1- Effective: Cidal (irreversible killing) vs. Static (reversible inhibition of growth & multiplication). 2- Kill microbes before they mutate & develop resistance. 3- Bioavailability: [ ] in specific body tissue for long enough time. 4- Selective toxicity (antimicrobials vs. disinfectants): toxic to & target microbial bacterial (1) cellular structures (penicillins/ peptidoglycan synthesis) or (2) metabolic pathways (sulphonamides/ folic acid synthesis) rather than normal human body cells. Highly selective antimicrobials are normally NOT toxic, however, they still can have side effects. 5- Side effects. 6- Stable in active form: upon storage in solid or liquid forms. 11-Nov-15 NM Kaplan 3
4 Doctor s Prescription (Rx) Name: scientific vs. trade. Dose: adult vs. child; healthy vs. unhealthy. Route of administration: 1- Local: Topical. 2- Systemic: PO, Parentral (IM, IV). Frequency: 1- Daily: (X4, X3, X2, X1). 2- Monthly. Duration: days, weeks, months. 11-Nov-15 NM Kaplan 4
5 Antibacterial Agents 1- Antibiotics: produced by soil living MOs: Moulds penicillin & cephalosporins. Bacteria bacitracin, erythromycin & chloramphenicol. 2- Semisynthetic: chemically modified to spectrum or side effects. 3- Synthetic. Activity spectrum 1- Narrow: targeting GP only as vancomycin. GN only as colistin & nalidixic acid. 2- Broad: target both GP & GN as ampicillin, chloramphenicol & tetracycline. 11-Nov-15 NM Kaplan 5
6 Categories of Antibiotics Antibiotics work most efficiently in conjunction with active IS to eradicate infecting bacteria in host: 1- Bactericidal (irreversibly kill S bacteria): serious lifethreatening infections of bone, endocardium & meninges. 2- Bacteriostatic (reversibly inhibit growth of S bacteria): NOT in immunocompromised, only in immunocompetent in D sufficient to allow cellular & humoral defense mechanisms to eradicate bacteria. Therapeutic Index = ratio of dose toxic to host to effective therapeutic dose toxic to bacteria. The higher the therapeutic index, the better the antibiotic. 11-Nov-15 NM Kaplan 6
7 Antimicrobial Susceptibility/ Sensitivity Testing (AST) CML processing of clinical specimens isolation of pure bacterial colonies Id & in-vitro AST to variety of antimicrobials. 11-Nov-15 NM Kaplan 7
8 AST Basic quantitative measures of in vitro activity of antibiotics: 1- Minimum Inhibitory Concentration (MIC) of antibiotic = lowest concentration inhibition of visible growth (as colonies on plate or turbidity in broth culture) under standard conditions. 2- Minimum Bactericidal Concentration (MBC) of antibiotic = lowest concentration killing of 99.9% of original inoculum in given time. Antibiotic s dose, route & frequency of administration absorption & distribution effective therapeutic dose (MIC or MBC) at site of infection. 11-Nov-15 NM Kaplan 8
9 Disk Diffusion test (Kirby-Bauer) Most common test for AST in CML as measure of antibiotic activity. Pure bacterial isolate suspension uniform inoculation onto entire surface of nutrient agar plate. Filter-paper disk impregnated with standard amount of antibiotic is applied to surface antibiotic s diffusion into adjacent medium gradient of antibiotic surrounding disk. After 16-24h incubation at 35 C under standardized conditions, bacterial semi-confluent growth appears zone of inhibition of bacterial growth around antibiotic disk is inversely related to MIC & its size is dependent on antibiotic diffusion rate and bacterial growth rate & degree of sensitivity. 11-Nov-15 NM Kaplan 9
10 Interpretation acc. to standard zones of inhibition for each antibiotic: 1- zone of inhibition standard MO is sensitive. 2- zone of inhibition < standard MO is resistant. 11-Nov-15 NM Kaplan 10
11 11-Nov-15 NM Kaplan 11
12 Combination/ Combined/ Multidrug Rx ( 2 agents with different modes of action simultaneously) 1- To kill all pathogens developing D course of ID & to prevent emergence of resistant mutants (e.g. TB). 2- For empirical (empiric) Rx. 3- To take advantage of antibiotic synergism. Antibiotic synergism when effects of dual combination of antibiotics > algebraic sum of effects of individual antibiotics. Antibiotic antagonism when effects of dual combination of antibiotics < algebraic sum of effects of individual antibiotics (< effect of most effective individual antibiotic when the other with least effect interferes with its effects e.g. Chloramphenicol & tetracyclines are considered to be antagonists to penicillins & aminoglycosides). 11-Nov-15 NM Kaplan 12
13 Empirical (Empiric) Rx For emergency IDs prior to final CML report, based on most likely pathogen (guess) & most effective drug acc to several considerations: 1- Lab report of pathogen Id refer to (Pocket chart = CML s accumulated AST D last year). 2- Pt s allergy. 3- Pt s age (NOT weight). 4- Pregnancy: Avoid Teratogenic. 4- In- or out-pt: IV drugs & Hospital formulary. 5- Pt s site of infection: Cystitis; [ ] in urine/ Brain abscess; cross BBB. 6- Pt s other medications: Drug cross reactions. 7- Pt s other medical problems: Toxicity. 8- Pt s immunocompromised status: Bactericidal. 9- Drug s cost: inexpensive narrow-spectrum>expensive, broadspectrum. 11-Nov-15 NM Kaplan 13
14 11-Nov-15 NM Kaplan 14
15 5 most common mechanisms of action of antibacterial agents 1- Inhibition of cell wall synthesis. 2- Damage to cell membrane. 3- Inhibition of protein synthesis. 4- Inhibition of nucleic acid (DNA or RNA) synthesis. 5- Inhibition of enzyme activity. 11-Nov-15 NM Kaplan 15
16 I. Inhibitors of bacterial cell wall (peptidoglycan/ murein/ mucopeptide) synthesis All are Bactericidal: cell wall synthesis inhibition cell wall enzymatic autolysis intracellular osmotic pressure rupture of inner &/or outer membranes release of contents cell death. 11-Nov-15 NM Kaplan 16
17 Transpeptidation (= peptide crosslinking) of peptidoglycan in w D-alanine is enzymatically excised from end of peptide side chain of existing cell wall polymer allowing it to be cross-linked by new peptide bond to recently synthesized peptidoglycan subunit. 11-Nov-15 NM Kaplan 17
18 11-Nov-15 NM Kaplan 18
19 11-Nov-15 NM Kaplan 19
20 11-Nov-15 NM Kaplan 20
21 11-Nov-15 NM Kaplan 21
22 -lactams include penicillins, cephalosporins, monobactam & carbapenems. All have in common 4-sided -lactam ring. Ppenicillins & cephalosporins have double-ringed structure with additional 5-sided ring & 6-sided ring respectively. Monobactam (Aztreonam) consist of -lactam ring alone with antibiotic activity. 11-Nov-15 NM Kaplan 22
23 Penicillins Maximum effect on actively dividing bacteria. Originally acid labile penicillin G active against: GPC (S. aureus, Strep. pneumoniae, Strep. pyogenes), GPB (B. anthracis, C. diphtheriae), GNC (N. gonorrhoea, N. meningitidis), anaerobic bacteria (Clost. perfringens) & spirochetes (T. pallidum). Other semisynthetic penicillins have stability (better absorption from GIT), elimination rates ( administration frequency) & wider spectrum. Many penicillins have little activity against GN bacteria as they do not penetrate outer membrane. Cephalosporins & other extended-spectrum newer penicillins are active against GN bacteria as they can penetrate outer membrane. 11-Nov-15 NM Kaplan 23
24 3 mechanisms of Resistance to -lactams 1- Bacterial -Lactamases (2 types): a- Penicillinases w destroy -lactam ring (Garage) in penicillins. b- Cephalosporinases w destroy - lactam ring in cephalosporins. 2- Chromosomal mutation change in PBPs structure inefficient binding of penicillins. 3- Chromosomal mutation in GN bacteria modified porins penicillins can NOT pass across outer membrane. 11-Nov-15 NM Kaplan 24
25 Polymyxin B binds preferentially to lipid-a of LPS & to phospholipids disruption of outer membrane of GN bacteria. has little activity against GP bacteria as their cell membrane is NOT exposed. can also lyse eukaryotic membranes selective toxicity toxic to human cells limited clinical use. 11-Nov-15 NM Kaplan 25
26 III. Inhibitors of Protein Synthesis mostly bacteriostatic. Selective toxicity: prokaryotic ribosome (70S=50S+30S subunits) vs. eukaryotic ribosome (80S=60S+40S subunits). 11-Nov-15 NM Kaplan 26
27 11-Nov-15 NM Kaplan 27
28 A. Antimicrobials w bind to 30S ribosomal subunit 1. Aminoglycosides (Bactericidal) Mode of action: irreversibly bind to 30S ribosome & freeze 30S initiation complex (30S-mRNA-tRNA) NO further initiation. Also slow down already initiated protein synthesis misreading of mrna. Broad spectrum: aerobic GN enterobacteriaceae, P. aeruginosa, V. cholera & S. aureus. Ineffective against anaerobes (as oxygen is required for uptake of antibiotic) & intracellular bacteria. Resistance: common. Synergy: with β-lactams w inhibit cell wall synthesis permeability to aminoglycosides. 11-Nov-15 NM Kaplan 28
29 2. Tetracyclines (Bacteriostatic) Mode of action: reversibly bind to 30S ribosome & inhibit binding of aminoacyl-trna to acceptor site on 70S ribosome. Broad spectrum: intracellular bacteria (chlamydias, rickettsias), mycoplasmas, V. cholerae & spirochetes (as Borrelia spp & T. pallidum). Resistance: common. Adverse effects: staining & impairment of structure of bone & teeth. Destruction of normal intestinal flora secondary infections (superinfection). 11-Nov-15 NM Kaplan 29
30 3. Spectinomycin (Bacteriostatic) Mode of action: reversibly interferes with mrna interaction with 30S ribosome. Structurally similar to aminoglycosides but does NOT cause misreading of mrna. Spectrum of activity: used in Rx of penicillin-resistant N. gonorrhoeae. Resistance: rare in N. gonorrhoeae. 11-Nov-15 NM Kaplan 30
31 B. Antimicrobials w bind to 50S ribosomal subunit 1. Chloramphenicol (bacteriostatic), Lincomycin (bacteriostatic), Clindamycin (bacteriostatic or bactericidal acc. to [drug] & bacterial sp.) Mode of action: bind to 50S ribosomal subunit & inhibit peptidyl transferase activity. Spectrum of activity: Chloramphenicol Broad. Lincomycin & clindamycin Restricted. Resistance: common (R to chloramphenicol by E w acetylates drug). Adverse effects: Chloramphenicol is toxic (BM suppression) used topically. 11-Nov-15 NM Kaplan 31
32 2. Macrolides (Bacteriostatic usually & Bactericidal at higher concentrations) Mode of action: bind to 50S ribosomal subunit & inhibit translocation of peptidyl trna. Spectrum of activity: GP bacteria in penicillin allergy, chlamydias, mycoplasmas, legionella spp. & T. pallidum. Resistance: common. Most GN bacteria are resistant to macrolides. 11-Nov-15 NM Kaplan 32
33 C. Antimicrobials w interfere with elongation factors Fusidic acid (Bacteriostatic) Mode of action: binds to elongation factor G (EF-G) & inhibits release of EF-G from EF-G/GDP complex. Spectrum of activity: only effective against GP bacteria as Streptococci, S. aureus & Corynebacterium minutissimum. 11-Nov-15 NM Kaplan 33
34 IV. Inhibitors of Nucleic Acid Synthesis Bactericidal. Selective toxicity: differences in prokaryotic & eukaryotic enzymes. 11-Nov-15 NM Kaplan 34
35 A. Inhibitors of RNA Synthesis Rifampin, rifamycin, rifampicin Mode of action: bind to DNA-dependent RNA polymerase & inhibit initiation of RNA synthesis. Spectrum of activity: broad but used most commonly in Rx of TB. Resistance: common usually used in combination Rx. 11-Nov-15 NM Kaplan 35
36 B. Inhibitors of DNA Synthesis 1- Quinolones Mode of action: inhibit bacterial DNA gyrase (topoisomerase) prevent DNA supercoiling. Spectrum of activity: cipro- (oral/ IV) is active against Enterobacteriaceae & Ps. aeruginosa & used in UTIs. Resistance: common for nalidixic acid & is developing for ciprofloxacin. 11-Nov-15 NM Kaplan 36
37 V. Antimetabolite Antimicrobials A. Inhibitors of folic acid synthesis (Bacteriostatic) Selective toxicity: bacteria cannot use pre-formed folic acid & must synthesize its own vs. mammalian cells w can use ready-made dietary folic acid. 11-Nov-15 NM Kaplan 37
38 11-Nov-15 NM Kaplan 38
39 B. Anti-Mycobacterial Agents Generally used in combination with other antimicrobials as Rx is prolonged & R develops readily to individual agents. In TB, 4 routinely prescribed drugs (isoniazid, rifampin, pyrazinamide & ethambutol) & 12 drugs required for R strains. 11-Nov-15 NM Kaplan 39
40 Undesirable/ side effects of antimicrobial agents 1- Selection for resistant organisms: M. tb, Plasmodium spp, S. aureus. 2- Allergy: low dose of penicillin G sensitization of atopic pts later second dose anaphylactic shock/ hives. 3- Toxicity: chloramphenicol aplastic anemia & streptomycin auditory nerve damage & deafness. 4- Prolonged oral broad-spectrum antibiotics destruction of indigenous microflora of mouth, intestine & vagina superinfection (overgrowth of opportunistic or 2ry invader MOs w are usually present only in small numbers e.g. Clos. difficile AAD & PMC and C albiacns vaginitis). 11-Nov-15 NM Kaplan 40
41 Drug Resistance (R) Superbugs = MDR Bacteria: 1- MRSA & MRSE (R to all anti-staph drugs except Vancomycin, Teicoplanin, Linezolid & Daptomycin), VISA (R to usual Vancomycin doses) & VRSA (R to highest practical Vancomycin doses). 2- VRE. 3- MDR-TB (R to 2 most effective first line drugs: isoniazid & rifampin) & XDR-TB (also R to most effective second line drugs: fluoroquinolones & at least one of the following: amikacin, kanamycin, capreomycin). 4- MDR strains of Acinetobacter baumannii, Pseudomonas spp., Stenotrophomonas maltophilia, Burkholderia cepacia & others. 5- -lactamase-producing strains of Strep. pneumoniae & H. influenzae. 6- Carbapenemase-producing Klebsiella pneumoniae (KPC): R to penicillins, cephalosporins, aztreonam, carbapenems & others. 7- CRE (carbapenem-resistant enterobacteriaceae). Viruses as HIV, HSV & Flu viruses. Fungi both yeasts & moulds. Protozoa as P. falciparum, T. vaginalis, Leishmania spp. & G. lamblia. 11-Nov-15 NM Kaplan 41
42 Definitions 1- Clinical Resistance: MIC of antimicrobial agent for particular bacterial strain exceeds that w is achieved with safety in vivo. 2- Cross Resistance: single mechanism of R to multiple antimicrobial agents & commonly seen with closely related antimicrobial agents. 3- Multiple resistance: multiple mechanisms of R are involved & seen with unrelated antimicrobial agents. 11-Nov-15 NM Kaplan 42
43 Mechanisms of bacterial resistance to antimicrobial agents Intrinsic R (Natural R): (e.g. cell-wall deficient mycoplasmas are R to inhibitors of cell wall synthesis & Strep/ Enterococcus are R to aminoglycosides w are unable to cross GP cell wall). Acquired R: S bacteria turns R to particular antibiotic by one of 4 mechanisms: 11-Nov-15 NM Kaplan 43
44 Strategies to drug resistance 1- Education of HCP & pts. 2- Justified prescription: of inexpensive narrow-spectrum effective antibiotic based upon clinical Dx, CML s results of culture & AST. 3- Complete prescribed therapeutic course: dose, frequency & duration. 4- Never use antibiotics for PPx (unless prescribed). 5- Good infection prevention & control procedures: hand washing, surveillance for alert MOs & isolation of pts infected with MDR. 11-Nov-15 NM Kaplan 44
45 Ignaz Semmelweis, Hungarian physician responsible for implementing hand washing practices & chlorine solutions in Obs ward in 1847 in his clinics (Vienna, Austria) & drastically reducing puerperal fever rates 11-Nov-15 NM Kaplan 45
46 Antiviral agents 11-Nov-15 NM Kaplan 46
47 Antifungal & antiprotozoal agents (fungi & protozoa are eucaryotic cells agents more toxic to host) Antifungal agents Bind cell membrane sterols (e.g. nystatin & amphotericin B). Interfere with sterol synthesis (e.g. clotrimazole & miconazole). Block mitosis or nucleic acid synthesis (e.g. griseofulvin & 5- flucytosine). Antiprotozoal agents Interfere with DNA & RNA synthesis (e.g. chloroquine, pentamidine & quinacrine). Interfere with protozoal metabolism (e.g. metronidazole). 11-Nov-15 NM Kaplan 47
48 Antifungal agents 11-Nov-15 NM Kaplan 48
49 11-Nov-15 NM Kaplan 49
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