University:
Franklin University
Course:
BIO-201 | Introductory Biology I
Academic year:

2020
Views:

103

Pages:

59
Author:

LunarLuminary

Antimicrobial Agents, Antimicrobial Resistance and Antimicrobial Susceptibility testing ANTIMICROBIAL AGENTS • Agents that kill or inhibit the growth of microorganisms. CLASSIFICATION 1. According to microorganisms against which they are used - antibacterial, antifungal, anti-parasitic, antiviral agents, etc. 2. According to their ability to kill or inhibit the microorganism- e.g. bactericidal and bacteriostatic. 3. According to the source – o Antibiotics o Chemotherapeutic agents CLASSIFICATION (cont..) 4. According to their site of action and usage• Disinfectants • Antiseptics • Antibiotics Mechanism of action of antimicrobial agents Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Drugs Spectrum of activity Mechanism of resistance Inhibit Cell Wall Synthesis Penicillin Penicillin G Aqueous penicillin G Procaine penicillin G Benzathine penicillin G Penicillin V Gram positive bacteria• Streptococcus pyogenes • Pneumococcus • Corynebacterium diphtheriae (diphtheria) • Clostridium tetani (tetanus) • Clostridium perfringens (gas gangrene) Others• Meningococcal infection • Gonococcus (penicillin resistance has been reported) 1. 2. 3. Drug inactivation (by producing β-lactamase enzyme) Alteration of target site Decreased permeability Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Drugs Spectrum of activity Mechanism of resistance Inhibit Cell Wall Synthesis Penicillinaseresistantpenicillins Methicillin Oxacillin Cloxacillin Dicloxacillin Same as penicillin plus Penicillinase producing Staphylococcus aureus Aminopenicillins (extended spectrum) Ampicillin Amoxicillin Same as penicillin plus • Enterococcus faecalis • Escherichia coli, Klebsiella • Helicobacter pylori • Salmonella (resistance reported) • Shigella (bacillary dysentery) 1. Drug inactivation (by producing β-lactamase enzyme) 2. Alteration of target site 3. Decreased permeability Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Drugs Spectrum of activity Mechanism of resistance Inhibit Cell Wall Synthesis Anti-pseudomonal penicillins Carbenicillin Ticarcillin Piperacillin Same as Aminopenicillins plus Pseudomonas aeruginosa β lactam + β lactamase inhibitors Aminopenicillins or Antipseudomonal penicillins plus • Clavulanic acid or • Sulbactam or • Tazobactam • Avibactam Same as aminopenicillins or anti-pseudomonal penicillins spectrum plus β lactamase producing bacteria 1. 2. 3. Drug inactivation (by producing βlactamase enzyme) Alteration of target site Decreased permeability Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Drugs Spectrum of activity Mechanism of resistance Inhibit Cell Wall Synthesis Cephalosporins 1st generation Cefazolin Cephalexin 2nd generation Cefoxitin Cefaclor Cefuroxime Ceftriaxone Cefotaxime Ceftazidime 3rd generation 4th generation 5th generation Cefepime Cefpirome Ceftobiprole Ceftaroline Staphylococcus aureus Staphylococcus epidermidis Some Gram-negatives like Escherichia coli & Klebsiella Same as 1st generation plus ↑ Gram-negative activity Decreased activity against Gram positives compared to the 1st and 2nd generation. ↑ Gram-negative activity Some are active against Pseudomonas (Ceftazidime) Good activity against Gram positive and negative bacteria including Pseudomonas Same as 4th generation plus Pseudomonas and MRSA (only β lactam to be ESBL (extended spectrum β-lactamases) Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Drugs Spectrum of activity Mechanism of resistance Inhibit Cell Wall Synthesis Carbapenems Imipenem Meropenem Doripenem Ertapenem Broadest range of activity against most bacteria, which include Gram positive cocci, Enterobacteriaceae, Pseudomonas, Listeria, anaerobes like Bacteroides fragilis and Clostridium difficile; except-MRSA, Mycoplasma etc. • Aztreonam Aztreonam Gram-negative rods Aerobes Hospital-acquired infections ESBL (extended spectrum β-lactamases) Produce carbapenamases. • Efflux pump Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Drugs Spectrum of activity Mechanism of resistance Other Cell Wall Inhibitors Glycopeptides (bactericidal: disrupt peptidoglycan cross-linkage) Fosfomycin Vancomycin Teicoplanin Active against most Gram positives MRSA (drug of choice), and indicated if the patient is allergic to β lactam drugs Alteration of target Fosfomycin 1. Alteration of target 2. Producing enzymes that inactivates fosfomycin Bacitracin Bacitracin Inactivates the enzyme UDP-Nacetylglucosamine-3enolpyruvyltransferase, also known as MurA; required for cell wall synthesis. Active against urinary tract pathogens; against both gram-positive and gram-negative bacteria such as E. faecalis, E.coli etc. Topical Gram-positive cocci infections Not defined Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Drugs Spectrum of activity Protein Synthesis Inhibition Anti-30S ribosomal subunit Aminoglycosides (bactericidal: irreversible binding to 30S) Gentamicin Neomycin Amikacin Tobramycin Streptomycin Aerobic Gram-negative bacteria Often used for empirical therapy in adjunct with third generation cephalosporins in respiratory infections, meningitis and subacute bacterial endocarditis Tetracyclines (bacteriostatic: bind to 30S subunit of ribosome and block tRNA attachment) Tetracycline Doxycycline Minocycline Demeclocycline Rickettsiae, Chlamydiae, Mycoplasma, Spirochetes Yersinia pestis, Brucella, Haemophilus ducreyi, Campylobacter Mechanism of resistance 1.Drug inactivation by aminoglycoside-modifying enzyme 2.Decreased permeability through Gram-negative outer membrane 3.Decreased influx of drug 1. Decreased intracellular drug accumulation (active efflux) 2. Ribosomal target site alteration Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Drugs Spectrum of activity Protein Synthesis Inhibition Anti-50S ribosomal subunit Haemophilus influenzae Pyogenic meningitis Brain abscess Anaerobic infection Enteric fever (Salmonella)- not used now due to development of resistance Mechanism of resistance Chloramphenicol Chloramphenicol Macrolides Erythromycin Azithromycin Clarithromycin Streptococcus Haemophilus influenzae Mycoplasma pneumoniae 1.Alteration of ribosomal target 2.Active efflux of antibiotic Lincosamide Clindamycin Bacteroides fragilis Staphylococcus aureus(toxic shock syndrome) Excellent bone penetration Alteration of target (ribosomal methylation) 1. Drug inactivation by producing chloramphenicol acetyltransferase enzyme 2. Altered membrane transport (active efflux) Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Drugs Spectrum of activity Protein Synthesis Inhibition Anti-50S ribosomal subunit Mechanism of resistance Linezolid Linezolid Resistant Gram-positives like MRSA Alteration of target site Streptogramins Quinupristin Dalfopristin Streptococcus pyogenes and Staphylococcus aureus skin infections MRSA infections VRE (Vancomycin resistant enterococci) infections 1. Alteration of target (dalfopristin) 2.Active efflux (quinupristin) 3.Drug inactivation (quinupristin and dalfopristin) Mupirocin Mupirocin Topical ointment is given for• Skin infections • Nasal carriers of MRSA Mutation of gene for target site protein Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Fluoroquinolones Drugs Fluoroquinolones 2nd generation Mechanism of resistance Nucleic acid Synthesis Inhibitors DNA synthesis inhibitors Inhibit DNA gyrase (A subunit) and topoisomerase IV, thus inhibiting DNA synthesis Nalidixic acid Fluoroquinolones 1st generation Spectrum of activity Coliform Gram negative bacilli Norfloxacin Ciprofloxacin Ofloxacin Levofloxacin Lomefloxacin Moxifloxacin Sparfloxacin Enterobacteriaceae- such as E.coli, Klebsiella, Enterobacter, Salmonella, Shigella, Proteus, Yersinia Others-Neisseria, Haemophilus, Campylobacter , Vibrio cholerae, Pseudomonas, Staphylococcus aureus 1. Alteration of target (mutation of DNA gyrase genes) 2.Poor transport across cell membrane Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Metronidazole Drugs Intracellularly generates metabolic by-products that damage DNA Spectrum of activity Nucleic acid Synthesis Inhibitors DNA synthesis inhibitors Anaerobic organisms Mechanism of resistance Not defined Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Drugs Spectrum of activity Mechanism of resistance RNA Synthesis Inhibitors Rifampin Inhibits RNA polymerase Staphylococcus Mycobacterium tuberculosis Mycolic Acids Synthesis Inhibitors Alteration of target (mutation of rpoB gene) Isoniazid Inhibits mycolic acid synthesis Tuberculosis Latent TB Mutations in enzyme processing isoniazid into active metabolites (KatG enzyme) Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Drugs Spectrum of activity Mechanism of resistance Folic acid Synthesis Inhibitors Bacteriostatic: Competitively inhibit enzymes involved in two steps of folic acid biosynthesis PABA(para-amino-benzoic acid) folate synthase Dihydrofolic acid dihydrofolate reductase Tetra-hydrofolic acid Sulfonamide blocks Trimethoprim blocks Sulfonamides and Trimethoprim Sulfadiazine Sulfacetamide Cotrimoxazole (Trimethoprim + Sulfamethoxazole) Sulfadiazine Used topically in burn wound surface Cotrimoxazole is indicated in• Urinary tract and respiratory tract infections- Active against Serratia, Klebsiella, Enterobacter • Shigella dysentery • Vibrio cholerae • Toxoplasma gondii • Haemophilus ducreyi • Pneumocystis jirovecii Production of insensitive targets that bypass metabolic block Antimicrobial agents; classification, indication and mechanism of resistance Class/ mechanism Drugs Spectrum of activity Antimicrobial agents that act on cell membrane Mechanism of resistance Gramicidin Forms pores Topical use against cocci (Gram positive and negative) Not defined Daptomycin Forms channels that disrupt membrane potential Polymyxin B (topical) Colistin or Polymyxin E (topical and systemic use) Bactericidal against Gram-positive bacteria including VRE and MRSA Not defined Polymyxins Gram-negative infections (disrupt both the outer and inner membranes in gram-negative cell wall) 1. Alteration of LPS 2. Efflux pump mediated ANTIMICROBIAL RESISTANCE • Antimicrobial resistance refers to development of resistance to an antimicrobial drug by a microorganism. • Can be of two types: o Intrinsic resistance. o Acquired resistance. INTRINSIC RESISTANCE • Refers to innate ability of a bacterium to resist a class of antimicrobial agents due to its inherent structural or functional characteristics Organism Anaerobic bacteria Aerobic bacteria Gram-negative bacteria Klebsiella species Pseudomonas Enterococci Serratia, Proteus and Burkholderia Stenotrophomonas maltophilia Intrinsic resistance against Aminoglycosides Metronidazole Vancomycin Ampicillin Sulfonamides, trimethoprim, tetracycline, or chloramphenicol Aminoglycosides All cephalosporins Polymyxin B and colistin Carbapenems ACQUIRED RESISTANCE • Emergence of resistance in bacteria that are ordinarily susceptible, by acquiring the genes coding for resistance • Overuse and misuse of antimicrobial agents is the single most important cause of development of acquired resistance. ACQUIRED RESISTANCE • Use of the particular antibiotic poses selective pressure in a population which in turn promotes resistant bacteria to thrive and the susceptible bacteria to die off. • Resistant bacterial populations flourish in areas of high antimicrobial use, where they enjoy a selective advantage over susceptible populations. ACQUIRED RESISTANCE • Factors favouring the spread of antimicrobial resistance includeo Poor infection control practices in hospitals- e.g. poor hand hygiene practices can facilitate transmission of resistant strains o Inadequate sanitary conditions o Inappropriate food-handling o Irrational use of antibiotics by doctors, not following antimicrobial susceptibility report o Uncontrolled sale of antibiotics over the counters without prescription. MUTATIONAL AND TRANSFERABLE DRUG RESISTANCE • In presence of selective antibiotic pressure, bacteria acquire new genes mainly by: o By mutation of resident genes. o By transferrable drug resistance-by acquisition of new genes by horizontal gene transfer mechanisms (conjugation, transduction, or transformation). Mutational resistance • Resistance developed due to mutation of the resident genes. • E.g. Mycobacterium tuberculosis – ATT • Low level resistance, developed to one drug at a time- overcome by using combination of different classes of drugs. Transferrable drug resistance • Transferrable drug resistance is plasmid coded - transferred by conjugation or rarely transduction, transformation. • Resistance coded plasmid (called R plasmid) can carry multiple genes, each coding for resistance to one class of antibiotic. • Results in high degree of resistance to multiple drugs, which cannot be overcome by using combination of drugs. Mutational vs transferable drug resistance Mutational drug resistance Transferable drug resistance Resistance to one drug at a time Multiple drug resistance at the same time Low degree resistance Resistance can be overcome by combination of drugs High degree resistance Cannot be overcome by drug combinations Virulence of resistance mutants may be Virulence not decreased lowered Resistance is not transferable to other organisms; but spread to off springs by vertical spread only Resistance is transferable to other organismsSpread by- horizontal spread (conjugation, or rarely by transduction /transformation) MECHANISM OF ANTIMICROBIAL RESISTANCE 1) Decreased permeability across the cell wall o Bacteria modify their cell membrane porin channels; either in their frequency, size, or selectivity. o Prevents antimicrobials from entering into the cell. o Observed in many Gram-negative bacteria such as Pseudomonas, Enterobacter and Klebsiella species against drugs such as imipenem, aminoglycosides and quinolones. MECHANISM OF ANTIMICROBIAL RESISTANCE 2) Efflux pumps o Mediate expulsion of the drug(s) from the cell, soon after their entry; thereby decreasing the intracellular accumulation of drugs. Observed in Escherichia coli and other Enterobacteriaceae against tetracyclines, chloramphenicol Staphylococci against macrolides and streptogramins Staphylococcus aureus and Streptococcus pneumoniae against fluoroquinolones MECHANISM OF ANTIMICROBIAL RESISTANCE 3) By enzymatic inactivation – o Inactivate the antimicrobial agents by producing various enzymes such as: β lactamase enzyme production - breaks down the β lactam rings, there by inactivating the β lactam antibiotics. Aminoglycoside modifying enzymes like (acetyltransferases, adenyltransferases, and phosphotransferases, produced by both Gram negative and Gram positive bacteria)  Chloramphenicol acetyl transferase MECHANISM OF ANTIMICROBIAL RESISTANCE 4)By modification of the antimicrobial target sites within the bacteriao Observed in:  MRSA -Target site of penicillin i.e. penicillin binding protein (PBP) gets altered to PBP-2a.  Streptomycin resistance in Mycobacterium tuberculosis- due to modification of ribosomal proteins or 16S rRNA.  Rifampicin resistance in Mycobacterium tuberculosis- due to mutations in RNA polymerase.  Quinolone resistance seen in many Gram positive bacteria, particularly S.aureus and S. pneumoniae - due to mutations in DNA gyrase enzyme. Beta Lactamase Enzymes • Hydrolyses the β- lactam rings (the active site) of β lactam antibiotics; there by deactivating their antibacterial properties. • Produced by both Gram positive and Gram negative organisms. • Plasmid coded, and transferred from one bacterium to other mostly by conjugation. β –lactamases classification 1. Ambler’s classification (structural or molecular classification)- According to this, β lactamases are classified into four classes. 2. Bush Jacoby Medeiros classification or functional (phenotypic) classification- This is the most advanced and complex classification. Ambler classification of beta lactamases Class A-ESBL(Extended spectrum β lactamases) Organisms producing ESBL enzymes are resistant to all Penicillins and 1st, 2nd& 3rdgeneration cephalosporins and monobactam, however remain sensitive to carbapenems and cephamycins • Resistance can be overcome by use of β lactam along with β lactamase inhibitor (e.g. sulbactum or clavulanic acid) • Detected by- Combination disc test (Ceftazidime and cetftazidime + clavulinic acid), Three dimensional test (best method) Class B-MBL (Metallobetalactamase) These organisms are resistant to all those antibiotics to which AmpC beta-lactamase producers are resistant plus they are resistant to carbapenems. • Resistance cannot be overcome by β lactam + β lactamase inhibitor combination • Detected by EDTA disc synergy test, modified Hodge test Ambler classification of beta lactamases Class C- AmpC beta-lactamase These organisms are resistant to all those antibiotics to which ESBL producers are resistant plus they are resistant to cephamycins (e.g. cefoxitin and cefotetan ). But they are sensitive to carbapenems. • Resistance cannot be overcome by β lactam + β lactamase inhibitor combination • Detected by AmpC disc test using cefoxitin disc Class D- oxacillinase Resistance can be overcome by β lactam + β lactamase inhibitor combination ANTIMICROBIAL SUSCEPTIBILITY TESTING • Bacteria exhibit great strain variations in susceptibility to antimicrobial agents – hence it is necessary to determine the susceptibility of pathogenic bacteria isolated from the clinical specimens. Classification of antimicrobial susceptibility testing methods 1. Disc diffusion methodso Kirby-Bauer disc diffusion method o Stokes disc diffusion method 2. Dilution testso Broth dilution method o Agar dilution method 3. E-test 4. Automated methods 5. Molecular methods (PCR detecting drug resistant genes) DISC DIFFUSION • • • • Most widely used method. Suitable for rapidly growing pathogenic bacteria. Unsuitable for slow growing bacteria. This method uses filter paper discs impregnated with appropriate concentration of the antibiotic solution. DISC DIFFUSION • Test bacterium is inoculated (as lawn culture) on the solid medium and then the antibiotic discs are applied. • The antibiotic in the discs diffuses through the solid medium • Concentration is highest near the site of application of the antibiotic disc and decreases gradually away from it. DISC DIFFUSION – Media (Mueller-Hinton agar) o Best medium to use for routine susceptibility testing of non-fastidious bacteria for the following reasons: It shows acceptable batch-to-batch reproducibility for susceptibility testing. It supports satisfactory growth of most non fastidious pathogens. It has minimal inhibitory effect on sulfonamide and trimethoprim. Hence these antibiotics are better tested in MHA than any other media. DISC DIFFUSION – Media (Mueller-Hinton agar) • Modifications of MHAo Lysed horse blood is added to MHA to support the growth of fastidious organisms such as H. influenzae. o 4% Sodium chloride (NaCl) should be added to the medium for testing MRSA isolates. DISC DIFFUSION – Inoculum preparation • Isolated pure colonies of the test organism are inoculated in a suitable liquid medium (peptone water broth). • Incubated at 35-37°C for 4-6 hours. • Density adjusted to 1.5 X 108cfu/ ml by comparing its turbidity with that of 0.5 McFarland opacity standard tube. • Ideal inoculum after overnight incubation gives even semiconfluent growth. Too heavy inoculum reduces the size of inhibition zones. DISC DIFFUSION – Lawn culture • Lawn culture • The following ATCC (American Type Culture Collection) strains are used as standard control strains. o Escherichia coli ATCC 25922 o Pseudomonas aeruginosa ATCC 27853 o Staphylococcus aureus ATCC 25923 o Enterococcus faecalis ATCC 29212 DISC DIFFUSION – Antibiotic disc • Antibiotic discs - 6 mm diameter are impregnated with standard quantity of antibiotic solution. • Choice of antibiotic disc : o Antibiotics should likely to be used for therapy of infections. o Local prescribing habits of the antimicrobial agents. o Resistant pattern of the locally prevalent pathogens o Cost, toxicity, pharmacokinetics, and spectrum of activity of an antimicrobial agent DISC DIFFUSION – Antibiotic disc • First line drugs o Commonly used for treatment. o Antibiotic currently being administered to the patient. o Spectrum should be relevant to the organism isolated. • Second line drugs o Restricted only to special circumstances. o Reserved for testing later if the organism is found to be resistant to all the first line antibiotics tested before. Kirby-Bauer Disc Diffusion Method • A cotton swab is dipped into inoculum and squeezed to drain out the excess fluid. • Swab is inoculated on to the Mueller-Hinton agar plate by lawn culture. • After drying the surface of agar plate for 3-5 minutes antibiotic discs are applied using either sterile forceps or multidisc dispenser. Kirby-Bauer Disc Diffusion Method • Plates are incubated at 37°C for 16-18 hours. • When tested for MRSA, result should be read only after 24hr of incubation. • Zone size is measured using a ruler or Vernier caliper. • Interpretation of zone size into sensitive, intermediate or resistant is based on the standard zone size interpretation chart. Commonly used disk concentrations and interpretation of disk diffusion test Antimicrobial agents Disc strength (µm) Benzyl penicillin (S.aureus) Cefoxitin (S.aureus) Gentamicin (G) Amikacin (Ak) Erythromycin (E) Tetracycline (T) Nitrofurantoin (Nf) Ciprofloxacin (Cf) Ceftriaxone (Ci) Imipenem (I) Vancomycin (for enterococcus) 10units 30 10 30 15 30 300 5 30 10 30 Diameter of zone of inhibition (in mm) Resistant Intermediate Sensitive ≤28 ≥29 ≤21 ≥22 ≤12 13-14 ≥15 ≤14 15-16 ≥17 ≤13 14-22 ≥23 ≤14 15-18 ≥19 ≤14 15-16 ≥17 ≤15 16-20 ≥21 ≤19 20-22 ≥23 ≤19 20-22 ≥23 ≤14 15-16 ≥17 Stokes Disc Diffusion Method • MHA plate is divided into three parts. • The test organism is inoculated on the central one third and the control strain on upper and lower thirds of the plate. • An Un-inoculated gap of 2-3 mm wide should separate the test and the control area on which the antibiotic discs are applied. • Plates are then incubated at 37°C for 16-18 hours. Primary Disc Diffusion Test • Performed when results are required urgently and single pathogenic bacterium is suspected in the specimen. • Disc diffusion directly performed from a clinical specimen. • Results of the primary test should be subsequently verified. DILUTION TEST • MIC (minimum inhibitory concentration) - Lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation. • Dilution tests: o Broth dilution o Agar dilution Broth Dilution • Serial dilutions of the antimicrobial agent in MH broth • Each tube is inoculated with a fixed amount of suspension of the test organism along with controls. • MIC is determined by noting the lowest concentration of the drug at which there is no visible growth. Broth Dilution • The minimum bactericidal concentration (MBC) can be obtained by subculturing from each tube (showing no growth) onto a nutrient agar plate without any antimicrobial agent. • The tube containing the lowest concentration of the drug that fails to show growth, on subculture, is the MBC of the drug for that test strain. • Broth dilution test can also be done using microtiter plates – micro broth dilution method. MIC – Clinical applications • For confirming the AST results obtained by disc diffusion tests. • For testing antimicrobial sensitivities of o slow growing bacteria such as tubercle bacilli. o bacteria for which diffusion test is not standardized • When a very small degree of resistance has to be demonstrated. • When the therapeutic dose of the drug has to be regulated accurately as in the treatment of bacterial endocarditis. Agar Dilution Method • Serial dilutions of the drug are prepared in molten agar and poured into petri dishes. • Test strain is spot inoculated. • Advantages or broth dilution o Several strains can to be tested at the same time by using the same plate o Directly measures the MBC; there is no need of sub culturing as it is done with broth dilution method. EPSILOMETER OR E-TEST • Uses an absorbent strip containing predefined gradient (serial dilution) of antibiotic concentration immobilized along its length. • Applied to a lawn inoculum. • Antibiotic concentration at which the ellipse edge intersects the strip, is taken as MIC value Automated Antimicrobial Susceptibility Tests • Automated systems are available: o VITEK 2 identification and antimicrobial sensitivity system (bioMerieux). o Phoenix System (Becton Dickinson) o Micro Scan Walk Away system • Principle - Micro broth dilution. MOLECULAR METHODS • Polymerase chain reaction (PCR) based assays are available targeting specific drug resistant genes • For example mecA gene for MRSA detection.