Exam 1 - Antibiotics Flashcards
Treatment
Antimicrobial agents are administered to cure existing or suspected infection
Antibiotic
- Substance used to prevent or treat infection caused by bacteria and other pathogenic microorganisms.
- Selectively inhibits a vital metabolic process of pathogens such as cell wall, DNA or protein synthesis.
- To be clinically useful, the compound needs to reach the site of infection at a sufficient concentration for an adequate length of time.
Prophylaxis
Antimicrobial agents are administered to prevent infection
Minimum inhibitory concentration (MIC)
Minimum concentration of antibiotic required to INHIBIT the visible growth of the test organism
Minimum bactericidal concentration
Minimum concentration of antibiotic required to KILL the test organism
(allows less than 0.1% of the original inoculum to survive)
Zone of Inhibition
Zone related to disk diffusion testing; a clear area surrounding an antimicrobial disk following overnight incubation; results from diffusion of the antimicrobial molecules into the agar and inhibition of growth of the test bacterium.
Bactericidal
Antimicrobial that kills a microorganism.
Or amount of antimicrobial agent required to kill.
Read any amount of growth in a microdilution MIC
For E-Test see picture
Bacteriostatic
Antimicrobial that inhibits bacterial growth but does not kill the bacteria.
Measured as ≥80% reduction in growth of the organism as compared to the control well.
For E-Test see attached picture
Example: Trimethoprim/sulfamethoxazole (SXT), Linezolid
Synergism
Occurs when the antimicrobial activity of a combination of antimicrobial agents is greater than the activity of the individual agents alone.
McFarland turbidity standard when preparing an inoculum for AST
0.5 McFarland standard represents 1.5X10 8 colony forming units (CFU/ml)
Susceptible (Sensitive) = S
An infection caused by the tested microorganism may be appropriately treated with the usually recommended dose of antibiotics.
Intermediate = I
The isolate may be inhibited by attainable concentrations of certain antibiotics (e.g., the beta-lactam antibiotics) if higher dosages can be safely used or if the infection involves a body site which allows the drug to concentrate (e.g., urinary tract).
This category serves as a buffer zone that prevents slight technical artifacts from causing major interpretative discrepancies. (Gray zone)
Resistant = R
Isolate is not inhibited by the concentration of antimicrobial agent normally achievable with the recommended dose, indicating specific resistance mechanisms are likely to be present.
Clinical and Laboratory Standards Institute (CLSI):
- Subcommittee of scientists and physicians
- Their goal is to establish standard conditions for testing methods based on laboratory investigations and assessment of clinical outcomes
- Updated guidelines are published annually for bacteria CLSI M100
What agar plate is used for setting up AST?
Mueller-Hinton
Quantitative AST Method
Broth, Agar dilution, or E-Test
Measures the Minimum Inhibitory Concentration (MIC) in micrograms per milliliter
Antimicrobial agents are usually tested at log2 (two-fold serial dilutions ex. 0.5, 1, 2, 4, 8, 16 etc.)
Qualitative AST Method
Agar diffusion (Kirby Bauer)
Categorizes an organism as susceptible (S), intermediate (I) or resistant (R) to a particular antimicrobial agent
Microbroth dilution method (<=0.1mL broth volume)
Quantitative
Indicates concentration of drug necessary to inhibit or kill the microorganisms tested
Doubling dilutions of antibiotic in broth
Turbidity visualization = MIC
50µl or 100 µl is directly dispensed into each of the 96 wells of the microtiter tray containing lyophilized antibacterial agents
The trays are examined for growth using a reflected viewing apparatus. The growth from broth alone is used as a comparison (positive control) .
The MIC is the lowest concentration that inhibits the visual growth of the organism
Advantages of Microbroth Dilution AST
- Reliable standardized method
- Convenient simultaneous testing of several antimicrobial agents against individual organisms
- Wide variety of products are commercially prepared; larger institutions can choose custom panels according to specific formulary needs
- Results of testing may be determined by visual exam or semi automated instrumentation
- It is considered a reference method in North America
Macrobroth dilution
(>=1 ml broth volume) – Very rarely performed in clinical labs.
Agar Dilution MIC
A series of plates containing various concentrations of each antimicrobial agent are prepared.
Test bacteria (0.5 McFarland) are spot-inoculated onto each plate using a multipronged replicating device.
After overnight incubation, the MIC is read as the lowest concentration of antimicrobial agent that inhibits the visible growth of the test bacterium
Currently considered the reference method for antimicrobial susceptibility testing of anaerobes and Neisseria gonorrhoeae.
Vitek 2 (for Automated Antimicrobial Susceptibility Testing)
This system facilitates standardized susceptibility testing in a closed environment with validated results and recognition of an organism’s microbial resistance mechanism in 6 to 8 hours for most clinically relevant bacteria.
Inoculum is automatically introduced via a filling tube into a miniaturized plastic 64-well, closed card containing specified concentrations (low, mid and high) of antibiotics.
Cards are incubated in a temperature-controlled incubator
Optical readings are performed every 15 minutes to measure the amount of light transmitted through each well and compared to the growth well without antibiotics.
Algorithmic analysis of the growth kinetics in each well is performed by the system’s software to derive the MIC data
The MIC results are validated with the Advanced Expert System (AES) software.
Disk Diffusion Method: (Kirby Bauer)
Allows categorization of most bacterial isolates as susceptible, intermediate or resistant to a variety of antimicrobial agents. (Qualitative)
The antibiotic diffuses from the disk, gradually decreasing in concentration as the distance from the disk increases.
At a critical point the amount of antibiotic is unable to visibly inhibit the organism being tested; thus, a zone of inhibition develops.
The point of this zone of inhibition is directly related to the MIC value.
Disk Diffusion Method: (Kirby Bauer) Procedure
- A standardized suspension of the organism is spread over the surface of an agar plate (Mueller-Hinton), within 15 minutes of preparation of the inoculum. The plate is swabbed in three directions to ensure even distribution
- Paper disks impregnated with antibiotics are placed on the agar surface within 15 minutes of inoculation of the agar plate. Disks should be placed at least 24 mm apart.
- Agar plates are incubated at 35°C within 15 minutes of set-up to prevent prediffuion of the antimicrobial agents into the agar
- Zone of complete inhibition is measured to the nearest millimeter
Disk Diffusion Method: (Kirby Bauer) Reading and Interpretation
The diameter of each inhibition zone is measured using a ruler or calipers. Plates are placed a few inches above a black, nonreflecting surface, and zones are examined from the back side (agar side) of the plate illuminated with reflected light.
Disk Diffusion results can be correlated to the MIC
The qualitative results of disk diffusion assay (KB) correlates well with quantitative results from MIC tests
Factors Affecting Size of Zone of Inhibition:
Inoculum density of organism
Larger zones with light inoculum and smaller zones with a heavy inoculum
Factors Affecting Size of Zone of Inhibition:
Timing of disk application
- The antibiotic disks must be applied to the media within 15 minutes after lawn of growth is made
- If after application of disk the plate is kept out longer than 15 minutes at room temperature, small zones may form
Factors Affecting Size of Zone of Inhibition:
Temperature of incubation
Larger zones are seen with temps less than 35degC
Factors Affecting Size of Zone of Inhibition:
Incubation time
Ideal 16-20 hours - less time does not give reliable results
Factors Affecting Size of Zone of Inhibition:
Depth of the agar medium
Thin media yields excessively large inhibition zones and vice versa
Factors Affecting Size of Zone of Inhibition:
Proper spacing of the disks
Avoids overlapping of zones
Factors Affecting Size of Zone of Inhibition:
Potency of antibiotic disks
Deterioration in contents leads to reduced size (possibly from condensation)
Factors Affecting Size of Zone of Inhibition:
Composition of medium
Affects rate of growth, diffusion of antibiotics and activity of antibiotics
Factors Affecting Size of Zone of Inhibition:
Reading of zones
Subjective errors in determining the clear edge
Gradient diffusion test (Epsilometer test “Etest”)
MIC is the point where the zone of inhibition intersects the strip
The strip has a gradient of antibiotics on it, place on bacterial lawn (0.5 McFarland), measure
Nitrocephin disks
Quick screen for beta-lactamase production
Nitrocephin is a cephalosporin
When you break open the beta lactam ring it changes color
Name and Recognize
Antimicrobial Targets and Mechanisms of Action (6)
1. Inhibition of Bacterial Cell Wall Biosynthesis
2. Inhibition of Folate Synthesis
3. Interference with DNA Replication
4. Interference with DNA Transcription
5. Interference of mRNA Translation
6. Combined Mechanisms of Action
Antimicrobial Targets and Mechanisms of Action
Inhibition of Bacterial Cell Wall Biosynthesis
Function by targeting bacterial cell wall synthesis. (β-Lactam antibiotics, such as penicillins, cephalosporins, carbapenems)
Vancomycin
Antimicrobial Targets and Mechanisms of Action
Inhibition of Folate Synthesis
- The folic acid pathway provides the essential precursor molecules needed for DNA biosynthesis in bacteria.
- Sulfamethoxazole (SMZ) blocks the step leading to the formation of 7,8-dihydropteroate.
- Trimethoprim (TMP) blocks the step leading to the formation of THF
- Example of folate pathway inhibitors: Sulfonamides and Trimethoprim
The spectrum of activity of folate pathway inhibitors, especially when provided in combination, provides activity against the Enterobacteriaceae that cause urinary tract infections.
Antimicrobial Targets and Mechanisms of Action
Interference with DNA Replication
- The prokaryotic cell cycle consists of DNA replication followed immediately by cell division.
- In microorganisms such as Escherichia coli, which divide in approximately 30 minutes under ideal growth conditions, DNA replication must be initiated and completed to ensure that each DNA duplex is delivered to each daughter cell.
- Enzymes necessary for DNA replication include topoisomerases I, II, III, and IV.
- Quinolones and fluoroquinolones (example: ciprofloxacin) are antibacterial drugs that affect DNA replication by targeting topoisomerases II (DNA gyrase) and IV.
The quinolones and fluoroquinolones are used to treat the Enterobacteriaceae, pseudomonads, Neisseria, and other gram-negative bacteria and staphylococci, enterococci, and streptococcal species other than Streptococcus pneumoniae.
Antimicrobial Targets and Mechanisms of Action
Interference with DNA Transcription
- DNA transcription is the process whereby a template DNA strand is copied into a functional RNA sequence, resulting in mature mRNA or structural RNA.
- Rifampin, a synthetic derivative of rifamycin B, targets DNA transcription. The principle therapeutic use of rifampin is in combination with other antibacterial classes to treat Mycobacterium tuberculosis infection.
Antimicrobial Targets and Mechanisms of Action
Interference of mRNA Translation
- Inhibits protein synthesis by targeting the ribosome (30s and 50s)
- The cellular machinery of living organisms decodes mRNA into functional protein, a process called mRNA translation. Protein biosynthesis requires the sequential binding of the 30S and 50S ribosomal subunits to mRNA, leading to translation of the genetic message.
- Because protein synthesis is central to cellular function, it is an excellent target for antibacterial drug product development. Thus the bacterial ribosome is a primary target of numerous drugs, with some targeting the 30S ribosomal subunit (e.g., aminoglycosides, tetracyclines, glycylcyclines) and others targeting the 50S ribosomal subunit (e.g., macrolides, lincosamides, chloramphenicol, oxazolidinones, streptogramins)
Penicillin binding proteins (PBP)
Penicillin binds to a variety of proteins in the bacterial cell membrane and cell wall, called penicillin-binding proteins (PBPs). Changes in PBPs are in part responsible for an organism becoming resistant to penicillin.
What are β-lactamases?
Penicillins (and cephalosporins) are called β-lactam drugs because of the importance of the β-lactam ring. An intact ring structure is essential for antibacterial activity; cleavage of the ring by penicillinases (β-lactamases) inactivates the drug.
Diagram where the β-lactamase enzyme cleaves the penicillin structure.
List examples of penicillinase-resistant penicillins
β-lactamase inhibitors (BLIs) clavulanic acid, sulbactam, and tazobactam.
What is the most common beta lactam/beta lactamase inhibitor combo?
Amoxicillin-clavulanic acid
Resistance to antimicrobial agents is divided into what 2 mechanisms?
Intrinsic and Acquired resistance
Define
Intrinsic Resistance
- Resistance that is naturally present in the microorganism. It is a property controlled by chromosomes and is related to the general physiology of the microorganism.
- Certain organisms will always be resistant in vivo to certain antibiotics regardless of how they test in vitro.
- Therefore this resistance is predictable once the organism is identified.
Describe 4 mechanisms of Intrinsic Resistance
- Biofilms (communities of microorganisms that are irreversibly attached to a solid surface)
- Impermeability (certain antibiotics unable to penetrate the cell wall of particular microorganisms)
- Efflux (function as transporter proteins for the extrusion of toxic substances and antimicrobial agents from the interior of the cell to the external environment.)
- Enzymatic Inactivation (produce enzymes that destroy the antimicrobial agents before they are able to reach their targets.)
K. pneumoniae is intrinsically resistant to what antibiotic?
Ampicillin
Enterococcus is intrinsically resistant to what class of antibiotics?
Cephalosporins
Acquired resistance
- Bacteria can utilize plasmids, transposons, and insertion sequences to transfer resistance genes to another bacterium.
- Can be expressed phenotypically as efflux, modification or acquisition of target sites, and enzymatic inactivation of the antibiotic.
- Acquired mechanisms of resistance are caused by changes in the usual genetic makeup of a microorganism, leading to altered cellular physiology and structure. Unlike intrinsic resistance, acquired resistance may be a trait associated with only some strains of a particular species. Thus the presence of this type of resistance in any of the isolates is unpredictable.
Describe Acquired Mechanisms of Resistance
- Efflux (some efflux pump genes have translocated to plasmids, which can be acquired by horizontal gene exchange.)
- Target Site Modification (Modification of a target can reduce the binding affinity of the antimicrobial agent for the target. Modification of target sites occurs primarily by chromosomal mutation) example: vanA gene for VRE
- Acquisition of New Targets (become resistant by acquiring cellular targets with reduced affinity for the antimicrobial agent.) example: mecA gene for MRSA
- Enzymatic Inactivation of Antimicrobial Agents (An existing cellular enzyme is modified to react with the antibiotic in such a way that it no longer affects the microorganism.) Example: CTX-M for ESBL
