Disease and Defense2.2 Flashcards
enterotoxin
a protein exotoxin released by a microorganism that targets the intestines.
endotoxin
also known as LPS, are large molecules consisting of a lipid and a polysaccharide composed of O-antigen, outer core and inner core joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria, and elicit strong immune responses in animals.
exotoxin
a toxin secreted by bacteria.
para-aminobenzoic acid or PABA
PABA is an intermediate in the synthesis of folate by bacteria, plants, and fungi. Many bacteria, including those found in the human intestinal tract such as E. coli, generate PABA from chorismate by the combined action of the enzymes 4-amino-4-deoxychorismate synthase and 4-amino-4-deoxychorismate lyase. Plants produce PABA in their chloroplasts, and store it as a glucose ester (pABA-Glc) in their tissues. Humans lack the enzymes to convert PABA to folate, so require folate from dietary sources such as green leafy vegetables. Although some intestinal bacteria can synthesize folate from PABA and some E. coli can synthesize folate, this requires six enzymatic activities in folate synthesis which are not all done in the same bacteria. In humans, PABA is considered nonessential and, although it has been referred to historically as “vitamin Bx”, is no longer recognized as a vitamin.Sulfonamide drugs are structurally similar to PABA, and their antibacterial activity is due to their ability to interfere with the conversion of PABA to folate by the enzyme dihydropteroate synthetase. Thus, bacterial growth is limited through folate deficiency
alanine racemase
this enzyme has one substrate, L-alanine, and one product, D-alanine. This enzyme participates in alanine and aspartate metabolism and D-alanine metabolism. It employs one cofactor, pyridoxal phosphate. At least two compounds, 3-Fluoro-D-alanine and D-Cycloserine are known to inhibit this enzyme. Bacteria can have one (alr gene) or two alanine racemase genes. Bacterial species with two genes for alanine racemase have one that is continually expressed and one that is inducible, which makes it difficult to target both genes for drug studies.
D-ala-D-ala pentapeptide
the two substrates of this enzyme are UDP-Mur2Ac(oyl-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala) and undecaprenyl phosphate, whereas its 3 products are UMP, Mur2Ac(oyl-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala)-, and diphosphoundecaprenol. This enzyme belongs to the family of transferases, specifically those transferring non-standard substituted phosphate groups. his enzyme participates in peptidoglycan biosynthesis.
transpeptidase
a bacterial enzyme that cross-links peptidoglycan chains to form rigid cell walls. In Gram-positive bacteria, the peptidoglycan molecules are cross-linked by a pentapeptide bridge, whereas, in Gram-negative bacteria, the peptidoglycan molecules are directly covalently bound to each other. The antibiotic penicillin irreversibly binds to and inhibits the activity of the transpeptidase enzyme by forming a highly stable penicilloyl-enzyme intermediate. Because of the interaction between penicillin and transpeptidase, this enzyme is also known as penicillin-binding protein (PBP).
Methicillin-resistant Staphylococcus aureus (MRSA)
MRSA is any strain of Staphylococcus aureus that has developed, through the process of natural selection, resistance to beta-lactam antibiotics, which include the penicillins (methicillin, dicloxacillin, nafcillin, oxacillin, etc.) and the cephalosporins. Strains unable to resist these antibiotics are classified as methicillin-sensitive Staphylococcus aureus, or MSSA. The evolution of such resistance does not cause the organism to be more intrinsically virulent than strains of S. aureus that have no antibiotic resistance, but resistance does make MRSA infection more difficult to treat with standard types of antibiotics and thus more dangerous.
vancomycin-resistant enterococci (VRE)
bacterial strains of the genus Enterococcus that are resistant to the antibiotic vancomycin. The mechanism of resistance to vancomycin found in enterococcus involves the alteration to the terminal amino acid residues of the NAM/NAG-peptide subunits, under normal conditions, D-alanyl-D-alanine, to which vancomycin binds. The D-alanyl-D-lactate variation results in the loss of one hydrogen-bonding interaction (four, as opposed to five for D-alanyl-D-alanine) being possible between vancomycin and the peptide. This loss of just one point of interaction results in a 1000-fold decrease in affinity. The D-alanyl-D-serine variation causes a six-fold loss of affinity between vancomycin and the peptide, likely due to steric hindrance.
Production of penicillinase enzyme via a plasmid
Induced in the presence of penicillin. May be transmitted by bacteriophages (transduction. Major problem with staphylococcus (MSSA). NOTE: β lactamase is generic term for enzymes that hydrolyze β-lactams for Penicillinases and Cephalosporinases
Alterations in penicillin-binding proteins
Responsible for methicillin resistance in staphylococci (MRSA)
Inability to penetrate into the bacterial cell in penicillins
Penicillin G can’t enter many gram neg bacteria (e.g., Pseudomonas) due to presence of outer membrane
“Escape” or Persisters Resistance to Penicillins
Metabolically inactive organisms or “L” forms can survive in a hypertonic environment like the kidney
penicillin absorption
Oral absorption varies depending on acid stability. Penicillin G poor and unreliable. Penicillin V and Amoxicillin excellent. Piperacillin and Ticarcillin and IV only. IM absorption dependent on salt form. Rapid from aqueous solutions. Delayed from suspensions (procaine – benzathine). Use against organisms susceptible to low but sustained levels of Pen G (syphilis- endocarditis)
Microbial toxins
are macromolecular products of microbes
that cause harm to susceptible animals by altering cellular structure or function. They are very potent, and the clostridial neurotoxins (botulinum and tetanus toxins) are the most toxic biological substances known. Some toxins cause the major manifestations of specific
diseases (for example: in botulism, cholera, diphtheria, whooping cough, scalded skin syndrome, scarlet fever, tetanus, or toxic shock syndrome). Other toxins contribute to pathogenesis without causing unique signs or symptoms (for example, pneumolysin). Toxin-mediated diseases cause significant morbidity and mortality, particularly in developing countries.
Traditional Methods to Show that a Specific Toxin Has a Role in Pathogenesis
Show that purified toxin causes the same symptoms or signs as infection by the toxin-producing microbe. Show that antitoxin prevents disease caused by the toxin-producing microbe. Show that virulence of individual bacterial strains correlates with the amount of toxin that they produce. Show that nontoxinogenic mutants are avirulent and that virulence is restored if the microbe regains the ability to produce toxin.
Molecular Version of Koch’s Postulates
Show that the phenotype or property to be investigated (e.g., toxin production) is associated with a pathogenic species or with pathogenic strains of a microbe. Show that inactivation of a specific gene(s) that encodes the putative virulence factor causes a measurable decrease in virulence of the microbe. Show that replacement of the mutated gene by the wild type allele restores virulence of the microbe to the original, wild type level.
Bacterial protein toxins
are usually heat-labile, immunogenic, and neutralized by specific antibodies. They were originally called “exotoxins” to indicate that they were found outside the bacterial cells. Some (like diphtheria toxin) are actually secreted into the culture medium. Others (like botulinum toxin) are released by lysis of the bacteria.
Lipopolysaccharides (LPS)
gram negative bacteria were first called “endotoxin” to indicate their association with bacterial cells. LPS is an example of a pathogen-associated molecular pattern (PAMP) that is recognized by the innate immune system and elicits host responses by a pathway that involves LPS binding protein, CD14, TLR4, and other signal transduction molecules. Low LPS doses activate macrophages, B-cells and the alternative complement pathway to cause fever, production of acute phase reactants, polyclonal antibody synthesis, and inflammation. High doses of LPS cause shock and disseminated intravascular coagulation. Many of the biologic effects of LPS are mediated by cytokines. LPS will not be discussed further in this lecture.
Toxins that facilitate spread of microbes through tissues.
Some toxic enzymes break down extracellular matrix or degrade debris in necrotic tissue (e.g., hyaluronidase, collagenase, elastase, deoxyribonuclease, and streptokinase), thereby enhancing spread of microbes.
Toxins that damage cellular membranes
Most membrane-damaging toxins kill target cells. Many are called hemolysins, because it is easy to detect their action on erythrocytes. Usually these toxins also damage other cells and are more accurately called cytolysins. Many membrane-damaging toxins insert into membranes and assemble into multimeric complexes that form pores, thereby causing lysis of target cells. Others, such as lecithinases, degrade specific cell membrane components and disrupt the integrity of the membranes.
Toxins that stimulate cytokine production
The pyrogenic exotoxins include erythrogenic (scarlatinal) toxins of Streptococcus pyogenes and the enterotoxins and toxic shock syndrome toxin (TSST-1) of Staphylococcus aureus. They are involved in scarlet fever, food poisoning, and toxic shock syndrome. The pyrogenic exotoxins belong to a larger class of molecules known as superantigens.
superantigens
are the most potent known T cell activators. They act by binding both to major histocompatibility (MHC) class II molecules on antigen-presenting cells and to specific Vβ chains on T cells at a site that is different from the antigen-binding site, and they activate much larger numbers of T cells than any specific antigen does. Superantigens stimulate excessive production of cytokines (including interleukin-2, interferon gamma, and others), thereby causing pathologic effects.
Toxins that inhibit protein synthesis
These toxins inhibit protein synthesis irreversibly and cause death of intoxicated host cells. Examples include diphtheria toxin, Pseudomonas aeruginosa exotoxin A, shiga toxins of shigella dysenteriase, and E coli.
Diphtheria toxin and Pseudomonas aeruginosa exotoxin A
inactivate elongation factor 2 (EF-2), which is required for peptide chain elongation. It is a toxin that inhibits protein synthesis. They are both ADP ribosyltransferases that transfer adenosine diphosphate ribose (ADP-ribose) from nicotinamide adenine dinucleotide (NAD) to a modified histidine residue called diphthamide on EF-2, thereby inactivating EF-2 in the cytoplasm.
Shiga toxins of Shigella dysenteriae and E. coli
and the plant
toxin ricin, are highly specific RNA N-glycosidases that remove one particular adenine residue from the 28S RNA of the 60S ribosomal subunit, thereby inactivating the ribosomes. It is a toxin that inhibits protein synthesis.
Toxins that modify intracellular signaling pathways
These toxins alter specific cellular functions and may or may not cause cell death. Examples include heat-labile enterotoxins of Vibrio cholerae and Escherichia
coli, Pertussis toxin, Heat-stable enterotoxin I (ST-I) of E. coli, Anthrax edema factor (EF), Anthrax lethal factor (LF), and Clostridium difficile toxins A and B.
Heat-labile enterotoxins of Vibrio cholerae and Escherichia
coli
are ADP ribosyltransferases that increase cell membrane-associated adenylate cyclase activity by ADP-ribosylating and activating the α subunit of the stimulatory Gs regulatory protein of the cyclase complex. Increased intracellular cAMP in small intestinal enterocytes causes active chloride secretion and results in secretory diarrhea. It is a type of toxin that modifies intracellular signaling pathways.
Pertussis toxin
an ADP ribosyltransferase that increases cell membrane-associated adenylate cyclase activity by ADP-ribosylating and inactivating the α subunit of the inhibitory Gi regulatory protein of the cyclase complex. Increased intracellular cAMP causes tissue- specific effects. It is a type of toxin that modifies intracellular signaling pathways.
Heat-stable enterotoxin I (ST-I) of E. coli
activates cell membrane-associated guanylate cyclase. Increased intracellular cGMP in enterocytes also causes secretory diarrhea. It is a type of toxin that modifies intracellular signaling pathways.
Anthrax edema factor (EF) from Bacillus anthracis and adenylate cyclase toxin from Bordetella pertussis
pertussis are adenylate cyclases that enter target cells, cause intracellular cAMP to increase, and produce cAMP-dependent effects. Their enzymatic activity requires activation by calmodulin and calcium, which are provided by the target cells. It is a type of toxin that modifies intracellular signaling pathways.
Anthrax lethal factor (LF)
an endopeptidase that cleaves several MAP kinase kinase proteins and inactivates their function in signal transduction. It is not yet know precisely how inactivation of this signal transduction pathway contributes to the lethal effects of LF. It is a type of toxin that modifies intracellular signaling pathways.
Clostridium difficile toxins A and B
are glucosyl transferases that alter the actin cytoskeleton of target cells by transferring glucose from UDP-glucose to several Rho family GTPases (including Rho, Rac and Cdc42), thereby inactivating them. It is a type of toxin that modifies intracellular signaling pathways.
Toxins that inhibit release of neurotransmitters
Includes botulinum toxin tetanus toxin.
Botulinum toxin
(7 antigenic types, A-G) causes flaccid paralysis of skeletal muscles by inhibiting release of acetylcholine at myoneural junctions. Types A, B and E most often cause disease in humans.
Tetanus toxin
(1 antigenic type) causes sustained muscular contraction (spastic paralysis/tetany) of skeletal muscles by inhibiting the release of neurotransmitter from inhibitory interneurons in the spinal cord.
Zinc dependent endopeptidases
Tetanus toxin and the botulinum toxins are zinc-dependent endopeptidases that inactivate specific SNARE proteins required for neuroexocytosis [VAMP (also called synaptobrevin), the 25 kDa synaptosome-associated protein (SNAP-25), and syntaxin]. Each toxin cleaves one specific protein at one specific site, and the individual serotypes of botulinum toxin differ in specificity from one another.
Therapeutic use for botulinum toxin
Botulinum toxin is used therapeutically to treat several focal dystonias and involuntary movement disorders, including strabismus and blepharospasm. It is also widely used for cosmetic effects.
Mechanisms and Pathways for Entry of Toxins into Cells
Toxins that act extracellularly or on plasma membranes are diverse
in structure and function. Their specificity is usually determined directly by the target of their action. Toxins with intracellular targets must cross the plasma membrane before they can exert their toxic effects.
Toxins with intracellular targets
They usually are bi-functional proteins with separate domains or subunits designated A (for active) and B (for binding). Susceptibility or resistance to toxins is often determined by presence or absence of receptors on the target cells. They typically use normal membrane constituents as receptors. Some toxin receptors are proteins: e.g., heparin-binding EGF-like growth factor precursor is the receptor for diphtheria toxin, and α2- macroglobulin receptor/low-density lipoprotein receptor-related protein [LRP] is the receptor for P. aeruginosa exotoxin A. Although diphtheria toxin and P. aeruginosa exotoxin A have identical intracellular actions, they act on different cell types because they utilize different cellular receptors. Some toxin receptors are glycolipids: e.g., ganglioside GM1 is the receptor for cholera toxin and E. coli heat-labile enterotoxins, and glycolipid Gb3 is the receptor for Shiga toxin. They usually enter target cells by endocytosis. Toxin receptor complexes enter endocytic vesicles and traffic to appropriate intracellular destinations. The active portion of the toxin is translocated to the cytosol to interact with its target. Diphtheria toxin, anthrax toxin protective antigen, botulinum toxin and tetanus toxin each have a translocation domain that changes its conformation in response to acidification of endosomes, inserts into the endosomal membrane to form a pore, and promotes translocation of the active component of the toxin to the cytosol. In contrast, Shiga toxin, cholera toxin, E. coli heat-labile enterotoxin, and pertussis toxin do not have a translocation domain. Instead, each of them trafficks via the retrograde pathway from endosomes, through the Golgi network, to the endoplasmic reticulum (ER). In the ER, the active component of each of these toxins is released from the holotoxin and is retro-translocated to the cytosol by a translocon of the ER-associated degradation (ERAD) pathway of the host cell. Subsequent intoxication of the cell results from the action of the active component of the toxin on its intracellular target.
Antitoxic antibodies (antitoxins)
bind to toxins and prevent their toxicity (neutralization). Antitoxins usually do not prevent infection by the toxin-producing bacteria or reverse toxic effects after the toxin has entered host cells.
Toxoids
are derivatives of toxins that retain immunogenicity but lack toxicity. They are used as vaccines for long term protection against toxin-mediated diseases.
Passive immunization
the administration of antibodies to a patient to provide immediate but temporary protection against a toxin or infectious agent. The duration of immunity is limited by degradation of the antibodies in the patient.
Active immunization
involves administration of toxoid to a patient in order to elicit production of specific anti-toxic antibodies. A primary series of immunizations and periodic booster doses are required to achieve and maintain protective levels of antitoxin. Active immunity can persist for many years because of immunologic memory.
Immunotoxins
“Immunotoxins” (and “hormonotoxins”) are hybrid molecules consisting of a toxin fragment that lacks the receptor-binding domain of the native toxin (derived, for example, from diphtheria toxin, exotoxin A, or ricin) and that is linked (by chemical conjugation or as recombinant fusion protein) to a ligand (such as a monoclonal antibody or a “single-chain” antibody [in the case of an immunotoxin] or a hormone or its receptor-binding domain [in the case of an hormonotoxin]) that exhibits binding specificity for a specific receptor than is different from the receptor for the native toxin. The rationale is enable the immunotoxin or hormonotoxin to bind to cells that express that alternative receptor and intoxicate them by delivering the toxic fragment of the native toxin. Many immunotoxins are designed to kill tumor cells that display a tumor-specific receptor but not to kill normal cells that lack that receptor. Immunotoxins are being tested as potentially valuable therapeutic agents for treatment of specific cancers, autoimmune diseases, and other disorders.
Mechanisms of Action in Antimicrobial Pharmacotherapy
The mechanism of antimicrobial drug action identifies the DRUG TARGET. Knowledge of the drug target provides insight into: The relative degree of Selective Toxicity, potential mechanisms for antimicrobial drug Resistance, and whether the mechanism is Bactericidal or Bacteriostatic
Pharmacokinetics of antimicrobial pharmacotherapy
Knowledge of absorption, distribution, and elimination (metabolism and excretion) aids in selection of the proper route and timing of administration of antimicrobial agent. Also allows for consideration of dosage adjustments when gastrointestinal / kidney / liver problems are present.
Cocci gram positive bacteria
Streptococci (pneumoniae, pyogenes, viridans), Staphylococci (aureus: MSSA vs MRSA), Enterococci (faecium, faecalis)
Cocci gram negative
Neisseria (gonorrheae)
Rods gram negative
H. influenzae, E. coli, Klebsiella, Pseudomonas aeruginosa
Anaerobes gram positive rod
Bacteroides fragilis
Mechanisms of action for antimicrobial chemotherapy
disrupting cell wall synthesis (vancomycin, bacitracin, penicillins, cephalosporins, monobactams, cerbapenems), folic acid metabolis (trimethoprim, sulfonamides), cell membrane (polymyxins and daptomycin), protein synthesis (tRNA) (mupirocin and linezolid), protein synthesis (30s inhibitors) (tetracycline, streptomycin, gentamicin, tobramycin (aminoglycoside), and amikacin), protein synthesis (50s inhibitors) (erythromycin (macrolides), chloramphenicol, clindamycin, and streptogrmins), DNA directed RNA polymerase (rifampin) an DNA gyrase (quinolones).
Inhibition of synthesis or damage to cell wall
: targets include stage 1 alanine racemase, stage 2 D-ala-D-ala pentapeptide, stage 3 transpeptidase.
Stage 1 alanine racemase
cycloserine; enolpyruvate transferase: fosfomycin
Stage 2 D-ala-D-ala pentapeptide
vancomycin; bactoprenol lipid carrier: bacitracin
Stage 3 transpeptidase
penicillins, cephalosporins, monobactams, carbapenems
Inhibition of synthesis or damage to cell membrane
targets are membrane components (daptomycin and polymixin B).
Modification of synthesis or metabolism of nucleic acids
targets include DNA gyrase, RNA polymerase, and DNA
Antibiotics targeting DNA gyrase
fluroquinolones
Antibiotics targeting RNA polymerase
rifampin
Antibiotics targeting DNA
metronidacole and nitrofurantoin
Inhibition or modification of protein synthesis
targets include 30s and 50s ribosome and Isoleucyl-tRNA synthetase
Antibiotics targeting 30S ribosome
aminoglycosides, tetracyclines
Antibiotics targeting 50S ribosome
clindamycin, macrolides, chloramphenicol, streptogramins
Antibiotics targeting Isoleucyl-tRNA-synthetase
mupirocin
Modification of intermediary metabolism
interrupts metabolism of folate, targets include dihydropteroate synthase and dihydrofolate reductase.
Antibiotics targeting Dihydropteroate synthase
sulfonamides
Antibiotics targeting Dihydrofolate reductase
trimethoprim
Selective toxicity of antibiotics
Fundamental feature of antibiotic therapy as the effects of antimicrobial agents should be exerted selectively on microbe and not the host. No “perfect” antibiotics exist. Biochemical differences between the pathogen target and the host must be discovered and appropriately exploited.
Inhibition of a metabolic pathway found in bacteria but not in humans
Folate metabolism: Bacteria must synthesize folate intracellularly, while mammalian cells
can take up folate from the environment
Pathway exists in both bacteria and humans, but differences in enzyme structure
Protein synthesis: Bacterial ribosome consists of 30S and 50S ribosome, while
mammalian ribosome subunits are 40S and 60S. Nucleic acid synthesis: DNA gyrase (bacteria) vs topoisomerase (humans); RNA
polymerase is structurally distinct in bacteria
Macromolecular structure does not exist in humans
Cell wall synthesis: Peptidoglycan component does not occur in eukaryotes
Macromolecular structure differs between microbes and humans
Fungal cell membrane: Ergosterol is the major constituent of fungal membranes vs cholesterol in mammalian membranes
Natural (intrinsic) Resistance
Microbes lack a susceptible target for drug action. E.g., fungal cell walls do not contain peptidoglycans and mycoplasma do not have cell walls at all, thus they are naturally resistant to penicillins. Pseudomonas aeruginosa is intrinsically resistant to many antibiotics because they cannot cross its outer membrane.
Escape resistance
Microbes are sensitive and antibiotic reaches target BUT organism “escapes” the consequences due to availability of purines, thymidine, serine, methionine released from purulent infections (sulfonamide resistance) or failure to “lyse” due to lack of osmotic pressure difference (penicillin resistance). Emphasizes important role for surgical drainage procedures if practical.
Acquired Resistance
Selective pressure (i.e., antibiotic administration) produces successive generations of organisms with biochemical traits that minimize drug action. Two modes: Mutational (chromosomal) resistance and Plasmid mediated resistance
Mutational (chromosomal) resistance
Plasmid mediated resistance
Plasmids are extrachromosomal pieces of circular DNA carrying genetic information
that can confer antibiotic resistance to the organism. This is a clinically important source of multiple drug resistance that can emerge
during a single course of treatment. Nonpathogenic coliform bacteria (gram negative) are a large reservoir for plasmid- mediated transfer of antibiotic resistance to pathogenic organisms; can code for resistance to multiple drugs (MDR gene) via protein that transports antibiotic out of cell. Exchange of genetic information among bacteria occurs by several mechanisms: Conjugation between two physically attached bacteria with exchange of plasmid DNA containing resistant determinant.
Transduction with virus (bacteriophage) carrying resistance determinant R to bacteria. Transformation - ability of certain bacteria to pick up free DNA from the environment.
Major mechanisms of bacterial resistance to antibiotics
includes altered targets, enzymatic destruction, alternative resistant metabolic pathway, decreased entry, and increased efflux.
Penicillin-binding proteins
[MRSA, S. pneumoniae, Enterococci) creates resistance in β-lactam antibiotics. Altered targets resistance or receptors to which the antibiotic cannot bind.
Altered DNA gyrase
[S. aureus, Pseudomonas species] can become resistance to Fluoroquinolones.
Peptidoglycan side chain alterations
[Enterococci (VRE), Staphylococci (VRSA)] leading to resistance against Vancomycin
50S ribosome methylation
[Strep-, Staph-, Enterococci] leading to resistance against Macrolides, Clindamycin
β-lactamase
produced by some bacteria that provide resistance to β-Lactam antibiotics like penicillins, cephamycins, and carbapenems (ertapenem). [S. aureus, P. aeruginosa, Bacteroides, Enterococci] leads to resistance against β-lactam antibiotics
Acetyl-/phospho-/adenylyltransferases
Enterococci] can become resistant to Aminoglycosides
Acetyltransferase
Staphylococci, Streptococci, Neisseria] can become resistant to Chloramphenicol
Overproduction of PABA or thymidine nucleotides
allows Streptococci to become resistant to Sulfonamides
Decreased entry (natural resistance)
β-lactam antibiotics [Pseudomonas aeruginosa] Fluoroquinolones [Pseudomonas species] Aminoglycosides [E. coli, Pseudomonas]
Increased efflux (multi-drug resistance may be encoded by single gene)
Tetracyclines [Streptococci, Staphylococci, Enterococci] Fluoroquinolones [Pseudomonas species]
Macrolides
Resistance can be minimized by
Only using antibiotic when need is established. Selecting antibiotic on basis of susceptibility tests. Using adequate concentration and duration to prevent emergence of first and second step mutants
Bactericidal vs. Bacteriostatic agents
Bactericidal (organisms are killed) vs bacteriostatic agents (organisms are prevented from growing). For a given antibiotic, whether or not it has a -cidal or a -static action is determined by its mechanism of action (target), concentration achieved in vivo, and the specific microorganism.
Bactericidal mechanisms
inhibition of cell wall synthesis, disruption of cell, disruption of cell membrane function, interference with DNA function synthesis. Bactericidal agent preferred in severe infections (assuming sensitive organism, drug distribution, and drug safety are compatible). Bactericidal agents act more quickly and their action is often irreversible with a sustained effect after the drug is eliminated from the blood. Bactericidal agents can compensate for patients with an impaired host defense (diabetes, agammaglobulinemia, immunosuppressive drugs, AIDS, asplenia). Bactericidal agents are required for treatment of infections in locations that are not accessible to host immune system responses (e.g., endocarditic vegetations and cerebrospinal fluid).
Bacteriostatic mechanisms
inhibition of protein synthesis (exception is aminoglycosides that end in –cidal) and inhibition of intermediary metabolic pathways.
Antimicrobial pharmacokinetics
Antimicrobial pharmacotherapy requires that the antibiotic reach the site of infection in sufficient concentrations (MIC) and once there remain active for a sufficient duration to be effective. Thus, in addition to a consideration of the pharmacodynamics of the antibiotic (antimicrobial activity against the specific infectious microorganism), it is also necessary to understand the pharmacokinetic properties: absorption from route (site) of administration, distribution to site of infection, elimination from body (hepatic or renal) as related to duration of antimicrobial activity.
Antimicrobial Absorption.
Provides information on the route of administration necessary for anti-infective effectiveness of the antibiotic. For most infections it is necessary to achieve adequate concentrations in the systemic circulation and most commonly this is accomplished via the oral or intravenous route. Certain infections can be managed with local application of the antibiotic, be it topically to the skin or mucous membranes for dermatological infections or oral administration of non-absorbable drugs for treatment of GI tract infections. The oral route has the advantage of ease of administration, patient acceptance, and lower cost while disadvantages can include GI upset or diarrhea due to alteration of intestinal flora, incomplete or lack of absorption for some drugs, and unsuitability in patients who are npo (nothing by mouth). “Take on empty stomach”: recommended when the antibiotic is unstable to the increased gastric acidity that occurs when food is in the stomach. “Take with food or meal”: recommended when the drug is acid stable but may be irritating to the stomach. The intravenous route is necessary for some drugs or some patients and has the advantage of providing the most rapid and predictable plasma levels when treating patients with life-threatening infections. Disadvantages associated with the IV route are the greater training needed, the greater expense, and the requirement for strict aseptic conditions. To reduce expense and complications seen with IV administration of antibiotics in hospitalized patients, efforts are being made to switch the patients to oral antibiotics whenever and as soon as possible (for certain antibiotics with excellent oral bioavailability [> 90%]).
Antimicrobial distribution of CNS
Most antibiotics distribute well to tissues outside the central nervous system, but vary substantially in their ability to cross blood brain barrier. Thus, penetration into the CNS is a necessary property of drugs that will be effective in treating CNS infections such as meningitis.
Antibiotics that readily enter the CSF
chloramphenicol, sulfonamides (trimethoprim), cephalosporins (3rd/4th), and rifampin (metronidazole).
Antibiotics that enter CNS with inflammation
penicillins, vancomycin, ciprofloxacin, and tetracycline.
Antibiotics that enter the CSF poorly
aminoglycosides, cephalosporins (1st/2nd), erythromycin, and clindamycin.
Antibiotics distribution to fetus
Adverse effects may occur in the fetus due to antibiotics that can cross the placental barrier. A general rule of thumb is that antibiotics that can be given orally, i.e., have the ability to cross the gastric mucosal barrier, can also cross the placenta and have the potential to harm the fetus. Drugs that should be used with caution or avoided during pregnancy include: aminoglycosides (D), chloramphenicol (C), fluroquinolones (C), metronidazole (1st trim), tetracyclines (D), and voriconazole (D).
Beneficial accumulations selective distributions/ accumulation
include clindamycin into bone (advantageous for treatment of osteomyelitis), concentration of macrolides into pulmonary cells (advantageous in upper respiratory infections), accumulation of tetracyclines into gingival crevicular fluid and sebum (advantageous in periodontitis and acne, respectively), rapid excretion of nitrofurantoin into urine (beneficial in urinary tract infections).
Selective accumulations that can increase the potential for toxicity
include aminoglycoside binding to cells of the inner ear and renal brush border resulting in an increased tendency for ototoxicity and nephrotoxicity or tetracyclines binding to Ca++ in developing bone and teeth resulting abnormal bone growth and brownish tooth discoloration in the fetus or young children.
Antimicrobial elimination
Knowledge of the route of antibiotic elimination is critical to the safe and effective use of antibiotics and at a minimum, one should know the major organ of elimination for antibiotics, either the kidneys or the liver.
Renal Excretion of Antimicrobial
Knowledge of those antibiotics drugs that are eliminated by renal excretion alerts
one to the possibility that renal dosing may be necessary in patients with kidney dysfunction. Renal dosing is the process whereby the dose and/or frequency of administration of the antibiotic are adjusted based on the patient’s renal function. Renal status is routinely monitored by measurement of serum creatinine (SCr) and estimation of creatinine clearance (CrCl).
Hepatic Metabolism of Antimicrobial
Knowledge of those antibiotics eliminated by drug metabolism alerts one to the possibility of metabolic drug-drug interactions, interpatient differences in metabolic rates (genetic polymorphisms), or hepatotoxic antibiotic actions. Unlike renally eliminated antibiotics, there is no lab value that gives a good estimate of the liver’s ability to metabolize antibiotics. Generally, antibiotics that require hepatic metabolism are simply avoided in patients with liver dysfunction if at all possible.
Duration of Antimicrobial Activity
Knowledge of the half-life of an antibiotic provides information regarding how often the drug will have to be administered to maintain the antimicrobial effect. Renal excretion and hepatic metabolism are the primary biologic processes that determine the plasma half-life of an antibiotic and the time that plasma levels remain above the MIC for a particular organism (its duration of action). Duration of therapy, along with the dose and dosage interval, can affect efficacy of therapy. If duration too short OR dose too low it can lead to resistance can develop as well as,
recurrence of infection. If duration too long, than superinfection more likely. If dose too high, than dose-related toxicities more likely to occur. For some antibiotics (aminoglycosides and fluoroquinolones), they continue to kill or inhibit growth of bacteria for several hours after the concentration of the drug falls below the MIC. This is known as the post-antibiotic effect and enables certain antibiotics to be given less frequently than would be predicted by their half-lives. Some antibiotics kill bacteria faster when given in doses that result in higher plasma concentrations, a property called concentration-dependent killing.
Clinical uses of antimicrobial
In hospitalized patients, culture and sensitivity results are often available within 24 hours, making the
selection of an antibiotic that will eradicate the microorganism causing disease less difficult. In the treatment of infections in the outpatient setting, culture and sensitivity data is infrequently available and antibiotics are commonly prescribed empirically based on symptoms, anatomic site, local
patterns of infections, and patient demographics.
In both settings, knowledge of antibiotic pharmacokinetics and toxicities is essential for selecting appropriate antibiotic for infection in a specific anatomic location in an individual patient.
Spectrum of Antibacterial Activity
The antibacterial spectrum of antibiotics can be imprecisely categorized into narrow spectrum (effective against either gram positive or gram negative), extended spectrum (effective against gram positive and gram negative), and broad spectrum (effective against gram positive, gram negative, and atypical organisms). Narrow spectrum antibiotics are often most effective on susceptible organism; less disturbance of host flora. Broad spectrum antibiotics can sacrifice efficacy for greater scope of activity for initial empiric coverage; more likely to cause superinfections. Acute severe infections should be treated with broad spectrum aggressive antibiotic therapy (target empiric therapy to likely pathogens and local antibiogram) with a switch to narrow spectrum coverage as soon as possible (target definitive therapy to known pathogens and antimicrobial susceptibility test results).
Antibacterial with narrow spectrums
aminoglycosides, penicillinase- resistant penicillins, clindamycin, vancomycin, metronidazole, penicillin G,V.
Antibacterial with extended spectrums
aminopenicillins (Amox-amp), cephalosporins, fluoroquinolones (cip, levo), carbapenemas.
Antibacterial with broad spectrum
macrolides, chloramphenicol, luroquinolones (moxi, gemi), sulfonamides, tetracyclines, trimethoprim.
Antimicrobial Drug Combinations
Indications for use include: Empiric treatment of severe infections of unknown etiology (until organism identified). Mixed infections [e.g., oral or intraabdominal infections]. Delay or prevent the emergence of resistant strains [e.g., tuberculosis and AIDS]. Obtain synergistic effect [e.g., penicillin plus an aminoglycoside against enterococci]
Direct Toxicity
Antibiotic effect on microbes affects host cellular processes (lack of selective toxicity). Varies with specific drugs and concentrations (at target and non-target sites). Can range from mild to
life-threatening. Usually involves GI tract (nausea and vomiting, diarrhea, pseudomembranous colitis), liver
(hepatotoxic metabolites, which can lead to liver failure), kidney (direct toxic effects on renal cells from drugs excreted by kidneys), nervous system (irritant properties and inherent neurotoxicity), blood and blood forming system (bone marrow depression).
Indirect Toxicity
Allergic reactions, hypersensitivity. “Salt” effects. Due to salt administered with the antibiotic, not the antibiotic itself (Na+, K+ salts of
penicillin, estolate salt of erythromycin). Drug-drug interactions. Antibiotics may alter activity of CYP450 drug metabolizing enzymes
Disturbances of Host Microflora
(Superinfection).Disturbances of ecological balance of microbial community. Allows overgrowth of normally
suppressed pathogenic organism. Pseudomembranous colitis due to Clostridium difficile overgrowth can be life-threatening. More commonly associated with broad spectrum antibiotics. Increased incidence noted if: age < 3 y/o
or > 50 y/o, pulmonary disease (non-tuberculosis), treatment of prolonged duration.
Host Factors
Age. Very old and very young have more susceptibility to toxicities. Pregnancy / nursing mothers. Need to also consider harmful effects on fetus or infant. Drug hypersensitivity. Patient allergies may preclude use of otherwise effective agent
Structure of penicillins
cysteine and valine condensation product. The is a thiazolidine ring that is fused to beta-lactam ring and side chain. Also has allergenic sulfur atom.
Production of penicillin
via fermentation. Semi-synthetic penicillins: Hydrolysis of side-chain with bacterial amidases to produce 6-amino-
penicillanic acid followed by chemical addition of side chains, using acyl chlorides. Compounds with different “R” groups have different properties including those that can:
Increase acid stability (in GI tract),
Decrease renal excretion, increase metabolic stability, Minimize bacterial resistance (β-lactamase or amidase), Increase antibacterial spectrum by increasing bacterial penetration. Early dosage of penicillin expressed in units. With advent of pure penicillins, this measure of penicillin quantity is outmoded. Used only for penicillin G. Amount of all other penicillins stated in mg. [1 mg Penicillin G = 1667 units OR 250 mg = 400,000 units]
Bacterial Cell Wall Synthesis
Stage 1: Synthesis and assembly of cell wall subunits occurring in the cytosol (inhibited by fosfomycin [1] and cycloserine [2]). Stage 2: Linear polymerization of subunits occurring at cell membrane (inhibited by bacitracin [3] and vancomycin [4]). Stage 3: Cross-linking of peptidoglycan polymers occurring at the cell wall (inhibited by penicillins, cephalosporins [5]). Conformation of penicillin is similar to that of D-ala-D-ala, the terminal portion of the pentapeptide of N-acetylmuramic acid peptide that cross-links with the pentaglycine portion of an adjacent N-acetylmuramic acid peptide.
Mechanism of action of penicillins
Penicillins are bactericidal to growing organisms. Lysis of spheroplasts depends on osmotic pressure difference between inner/outer environments. Satisfactory explanation for lysis is lacking. Action of Penicillin G mainly confined to gram-positive organisms and gram-negative cocci, some spirochetes. Acts on gram-negative bacilli, but much higher concentrations required. Penicillin Binding Proteins (PBPs): β-lactam antibiotics have a complex mechanism of action. They acylate several bacterial proteins
termed penicillin binding proteins (PBPs). PBPs include but are NOT LIMITED to transpeptidase enzymes (D-alanine carboxypeptidase,
endopeptidase). Penicillins inhibit these enzymes by an irreversible covalent interaction. Particular β-lactam antibiotics bind to distinct PBPs. Binding is NOT uniform. The antimicrobial spectrum of action of certain β-lactam antibiotics is distinctive and related to their binding to some but
not all PBPs. Autolytic Activity. The presence of endogenous autolytic enzymes (murine hydrolases) is required for
bactericidal lytic effect. β-lactams “trigger” this activity by depressing the natural inhibitory action of
autolysins. Kinetics of penicillin bactericidal action. Penicillin may also activate autolytic action of enzymes.
Relevance of PBPs to clinical use
Effect persists when drug is gone as penicillin exerts a persistent injurious action due to its
covalent binding to bacterial proteins. Maximal killing rate is function of the growth rate of the organism
Resistance to penicillin
Production of penicillinase enzyme via a plasmid. Production of the β-lactamase is induced in the
presence of penicillin. May be transmitted to sensitive organisms by bacteriophages (transduction). Major problem with staphylococcus. NOTE: β lactamase is the generic term for enzymes that hydrolyze β-lactams, includes penicillinases and cephalosporinases. Alterations in penicillin-binding proteins. Responsible for methicillin resistance in staphylococci (MRSA) and penicillin resistance in pneumococci. Inability to penetrate into the bacterial cell. E.g., penicillin G can’t enter many gram-negative bacteria (e.g., Pseudomonas) due to presence of outer membrane. “Escape or Persisters. Metabolically inactive organisms or “L” forms can survive in a hypertonic environment like the kidney.
Absorption of penicillin
Penicillins are moderately strong acids. Highly water-soluble. Acid-lability impairs oral absorption of many types of penicillin (penicillin G [about 20%], methicillin, carbenicillin, ticarcillin). Thus, optimal absorption from an empty stomach (1 hr ac or 2 hrs pc). Oral doses must be much higher than parenteral doses for penicillin G. Chemical modification of R-group improves absorption by increasing acid stability (amoxicillin, penicillin V). Rapidly absorbed from IM parenteral sites. Use of insoluble salts to reduce absorption and extend duration of action. For example, procaine penicillin G and benzathine penicillin G.
Distribution of penicillin
Distribute throughout body. Penetrate into tissues poorly (largely ionized at physiological pH).
Highest concentration in liver, kidney, skin. Variable binding to plasma proteins. Can enter inflamed tissues or membranes (CSF, joint, eye) more readily than normal.
Metabolism – Excretion of penicillin
Most penicillins excreted as active drug via the kidney (t1/2 < 1 hr).
90% by tubular secretion. Blocked by probenecid (can prolong duration of activity). Metabolism often increases to compensate in cases of renal failure (oxacillin-type). Excreted in breast milk (consider risk / benefit in use).
Individual penicillins
includes prototype, penicillinase- resistant, extended spectrum and β-Lactamase Inhibitors.
Prototype Penicillins
Relatively narrow spectrum of antimicrobial activity. Includes penicillin G and acid resistant penicillin.
Penicillin G
(Benzyl penicillin) [Pen G Benzathine: Bicillin, Permapen, Pen G Procaine: Wycillin,
Crysticillin]. The prototypical penicillin. Powerful and inexpensive. Previously penicillin of choice in most circumstances, but use today limited to hospitalized patients with serious infections given via the parenteral route. However, hydrolyzed by acid and penicillinase enzyme. About 30-50% bound to plasma protein.
Acid resistant penicillin
Penicillin V (Phenoxymethyl penicillin) [Pen Vee K, V-Cillin]. Better absorbed than penicillin G, but still incompletely absorbed. Many prefer Penicillin V for oral therapy because of higher reliability of absorption. Antimicrobial efficacy generally less than penicillin G but still suitable for many mild-to-moderate infections.
Penicillinase-Resistant Penicillins
In general order of efficacy (greatest first, but efficacy will vary depending on the particular organism): Methicillin [obsolete] > Nafcillin [Unipen (parenteral, erratic oral)] > Oxacillin [Prostaphlin, Bactocill (oral)] > Cloxacillin [Tegopen(oral)] and Dicloxacillin [Dynapen, Veracillin (oral)]. Considerably less potent against Penicillin G-sensitive organisms. Not substitutes for penicillin G, except when penicillinase-producing organisms are encountered (but emergence of MRSA has greatly limited current clinical use). Variable protein binding, high with oxacillin, cloxacillin and dicloxacillin. NOTE: Acid resistance varies among the penicillinase resistant penicillins as noted above. These penicillins are less susceptible to β-lactamase than are the cephalosporins. As a group the isoxazole penicillins are eliminated by both renal and hepatic routes improving their safety profile in patients with renal insufficiency. All are relatively narrow spectrum agents: Gram-positive, gram-negative cocci.
Extended Spectrum Penicillins
Increased hydrophilicity [due to presence of amino (NH2) or carboxyl (COOH) groups] allowing penetration through porins of outer membrane of gram-negative organisms.
Ampicillin [Omnipen] and amoxicillin [Amoxil]
they are extended spectrum penicillin. Possess significant additional activity against gram-negative bacilli. Acid resistant, but NOT resistant to penicillinase. Amoxicillin is more completely absorbed after oral administration and food interferes less with its absorption (advantage of less frequent dosing and less diarrhea). Not resistant to penicillinase, but both can be given with β- lactamase inhibitors to further extend their antimicrobial spectrum.
Anti-pseudomonal penicillins
(not resistant to penicillinase). Ticarcillin [Ticar] and Piperacillin [Pipracil]: Must be given parenterally. Effective against Pseudomonas aeruginosa and enterococci (often combined with
aminoglycosides). Also useful in anaerobic infections caused by Bacteroides fragilis.
β-Lactamase Inhibitors
Clavulanic Acid, Sulbactam, Tazobactam. Another pharmacologic approach to combat penicillin resistance. Resemble β-lactam molecules but have weak or no antibiotic activity. They act as potent, irreversible inhibitors of β-lactamase. Most active against plasmid-encoded β-lactamases. The β-lactamase inhibitor will extend the antibacterial spectrum of the accompanying penicillin only if bacterial resistance is due to β-lactamase destruction and the inhibitor is active against that particular β- lactamase. Clavulanic acid combined with amoxicillin (Augmentin, oral) and ticarcillin (Timentin, parenteral), sulbactam combined with ampicillin (Unasyn, parenteral), and tazobactam combined with piperacillin (Zosyn, parenteral).
Penicillin for gram positive cocci
Streptococci causes pharyngitis, pneumonia (CAP), sinusitis, otitis media, rheumatic fever, necrotizing fasciitis [Pen G, Pen V, Amoxicillin].
Enterococci causes bacteremia [Pen G, Ampicillin plus AG], urinary tract infection. [Ampicillin]. Staphylococcus aureus causes localized cutaneous infection, bacteremia, device-associated infections, pneumonia [MSSA: Oxacillin, MRSA: NO Penicillins].
Gram-negative cocci with penicillin
Neisseria gonorrhea causes gonorrhea [Pen G, but high levels of resistance].
Neisseria meningititis causes meningitis [Pen G].
Moraxella catarrhalis causes otitis media, community-acquired pneumonia [Amoxicillin - Clavulanate].
Gram-positive bacilli with penicillin
Bacillus anthracis causes anthrax [PenG].
Cornyebacterium diphtheria causes diphtheria [PenG].
Gram-negative bacilli with penicillin
[Ampicillin, Amoxicillin +/- Clavulanate]
H. influenzae causes meningitis, otitis media, sinusitis, pneumonia (CAP).
E. coli causes urinary tract infections, intra-abdominal infections, diarrhea, hemorrhagic colitis.
Klebsiella causes urinary tract infections, intra-abdominal infections.
H. pylori causes peptic ulcer disease.
Salmonella species causes gastroenteritis, typhoid fever.
Pseudomonas aeruginosa causes opportunistic infections in any organ or tissues [Amoxicillin-Clavulanate, Piperacillin - Tazobactam, Ticarcillin - Clavulanate].
Anaerobes
with penicillin
Clostridium perfringens (gram + rod) causes gas gangrene, food poisoning [Pen G]. Bacteroides fragilis (gram – rod) causes intraabdominal and brain abscess [Piperacillin - Tazobactam, Ticarcillin - Clavulanate].
Spirochetes with penicillin
Treponema pallidum causes syphilis [PenG]. Borrelia burgdorferi causes Lymedisease (early) [Amoxicillin].
Toxicity and Adverse Reactions with penicillin
Virtually non-toxic, except for hypersensitivity reactions.
Hypersensitivity Reactions with penicillin
Penicillin presumably functions as a hapten, combining with protein to form a complete antigen. Most reactions related to 6-aminopenicillanic acid portion of molecule; thus cross- sensitization occurs among penicillins. Classified by time to develop and immunologic type. IMPORTANT to note: 10% of patients report a penicillin allergy. Only 10-20% of these patients actually experience a reaction of some kind on exposure to a penicillin (1-2% overall). Classification of penicillin reactions are Type I, II, III
and IV. Other reactions are idiopathic, exact immunological mechanisms unknown. Most common reaction (1-4%) is a maculopapular or morbilliform rash that is generally mild and reversible.
Type I Reactions with penicillin
Very rare (0.05%), but potentially life threatening. Mediated by IgE (mast cells) antibodies: Urticaria, angioedema, respiratory obstruction, vascular collapse. Onset ranges from a few minutes to about 30 minutes.
Type II Reactions with penicillin
Rare, due to cytotoxic antibodies of the IgG or IgM class. Complement-dependent cell destruction; e.g. hemolytic anemia.
Type III Reactions with penicillin
Delayed allergic reactions (> 72 hours). Formation of IgG or IgM immune complexes with penicillin that act as antigens and can activate complement and lodge in tissue. Skin rashes, serum sickness, arthralgias, allergic vasculitis (inflammation of vascular bed).
Type IV Reactions with penicillin
Cell mediated allergy, delayed reactions. Mediated by T-lymphocytes. Skin eruptions, thrombocytopenia.
Other Toxicities than hypersensitivity with penicillin
Rarely, encephalopathy or seizures can occur with intrathecal penicillin or massive parenteral doses of
penicillin, especially if associated renal insufficiency. Beware of potassium or sodium intoxication when massive doses of salts and penicillin are administered
parenterally. Especially a problem if renal insufficiency or CHF. Jarisch-Herxheimer reaction during treatment of syphilis (via spirochetal antigens).
Vancomycin
[also synthetic lipoglycopeptide derivative – Telavancin (Vibativ)
Structure / Mechanism of Action of Vancomycin
Tricyclic glycopeptide acts by inhibiting cell wall synthesis at site
different from penicillin (blocks linear polymerization, Stage 2 of cell wall synthesis).
Pharmacokinetic Properties of Vancomycin
Poor oral absorption, administered IV , except for GI tract indications (e.g., Clostridium, but
metronidazole is now preferred agent due to resistance concerns and expense of vancomycin). Excretion mainly through kidneys, in renal failure half-life extended to 6-10 days.
Antimicrobial Spectrum / Clinical Uses
Use reserved for situations when less toxic agents are ineffective or not tolerated (e.g., penicillin allergy).
Gram positive cocci with Vancomycin
Methicillin Resistant Staphylococcus Aureus (MRSA) causes severe skin and soft tissue infections. Staphylococci and streptococci causes meningitis, pneumonia, endocarditis, sepsis.
Enterococci (ampicillin resistant) causes bacteremia, endocarditis.
Anaerobes with vancomycin
Clostridium difficile causes pseudomembranous colitis [vancomycin is used orally - parenteral route does not deliver to GI tract]; metronidazole 1st choice if NOT severe infection
Adverse Reactions with vancomycin
Chills-fever-skin rash (infusion-related), ototoxicity most severe (pretreat with acetaminophen and diphenhydramine). More highly purified preparations now available display fewer adverse effects.
