Abstract
Antimicrobial agents are used to kill or suppress the growth of microorganisms and are used widely both to treat and prevent infection. In order to understand how antimicrobial drugs work it is necessary to understand the anatomy and structure of microorganisms.
Antimicrobial agents are used to kill or suppress the growth of microorganisms and are used widely both to treat and prevent infection. In order to understand how antimicrobial drugs work it is necessary to understand the anatomy and structure of microorganisms.
Structure and Classification
Microorganisms capable of causing disease in humans consist of five main classes:
Bacteria
Viruses
Fungi
Protozoa
Algae.
When considering the cellular structure of macro and microorganisms there are two broad types, eukaryotes and prokaryotes (see Table 23.1). Cells making up animals, plants, fungi, protozoa and algae are eukaryotic. Characteristics of eukaryotic cells are that they are larger, more complex and have larger internal volumes. Prokaryotic cells are smaller, simpler and of a smaller internal volume. Bacteria are prokaryotes. The differences between mammalian cells and those of microorganisms are important for identifying therapeutic targets. These are important so that antimicrobial agents offer selective toxicity (toxicity to microorganisms without toxicity to host cells).
Bacteria
Morphology
Staining properties
Aerobic or anaerobic metabolism.
Bacteria have peptidoglycan cell walls. Bacteria are described in terms of their morphology (cocci, rods etc.) and formation of colonies (clusters, chains etc.). They are also classified by the staining pattern of their cell walls into three separate groups. This is directly relevant to the antimicrobial prescriber because the cell wall staining pattern guides the choice of antibiotic. The gram staining system relies on the differing staining properties of cell walls.
1. Gram-positive bacteria retain the initial dye crystal violet and appear purple under the microscope. They have a thick peptidoglycan cell wall with no additional outer membrane covering it (see Figure 23.1).
2. Gram-negative bacteria have a thin peptidoglycan cell wall, covered by an additional outer membrane rich in lipopolysaccharides (LPS), therefore they do not retain crystal violet dye. Gram-negative cells stain pink with the counter stain sufenin. When gram-negative bacteria break down upon death, they release large quantities of LPS into the circulation provoking an extreme cytokine response. LPS is also known as endotoxin.
3. Acid fast bacilli resist decolouration with the above stains. They retain red dye after contact with an acid/alcohol mixture.
Aerobic bacteria require oxygen to survive. Anaerobic bacteria do not. The aerobic or anaerobic nature, combined with the gram staining properties above, can be used to classify virtually all bacteria (see Table 23.2).
| Gram-positive | Gram-negative | |||
|---|---|---|---|---|
| Cocci (spheres) | Bacilli (rods) | Cocci (spheres) | Bacilli (rods) | |
| Aerobic | Staphylococcus S. aureus;S. epidermidisStreptococcusS. pneumoniaeEnterococcus E. faecalis | Bacillus B. anthracis |
|
|
| Streptococcus viridians |
|
| |
Antibiotics and Antimicrobial Chemotherapeutic Agents
Antibiotics are naturally occurring substances produced by one microorganism that act against another microorganism. Antimicrobial chemotherapeutic agents are synthetic substances that act against microorganisms but, for the purposes of this chapter when acting against bacteria, will be called by the common name, antibiotics.
Antibiotics can be classified by their:
mechanism of action (or more simply by whether they kill [bactericidal] or inhibit growth [bacteriostatic] of bacteria), or
pharmacological structure.
Therapeutic Targets for Antibiotics
Antibiotics target four different aspects of the microorganism in order to achieve their goal (see Figure 23.2).
Figure 23.2 Summary of the mechanism of action of antibiotics.
1. Inhibition of cell wall synthesis
• β-lactams
• Glycopeptides
Gram-positive bacteria rely on their thick cell walls for cellular integrity. They have no protective outer membrane or lipopolysaccharide layer; therefore, they are more susceptible to drugs affecting cell wall synthesis than Gram-negative bacteria.
2. Inhibition of protein synthesis
• Aminoglycosides
• Tetracyclines
• Macrolides
• Oxazolidinones.
3. Disruption of the cytoplasmic cellular membrane
• Polymyxins
• Colistins
• Daptomycin.
4. Inhibition of normal nucleic acid replication
• Fluroquinolones
• Sulphonamides
• Rifampicin
Pharmacokinetics of Antibiotics
In order to be effective, antibiotics must reach the site of infection which often involves passing across the capillary membrane and out of the circulation. Their ability to penetrate the infected tissue depends on local factors such as tissue perfusion and the nature of the barrier (e.g. blood brain barrier) and the properties of the antibiotic itself such as protein binding, lipid solubility and pKa. The organs with the greatest perfusion are the brain, kidney, liver, and heart and it would be expected that drug concentrations would increase most rapidly in these organs. Conversely, tissues with inherently poor blood flow (tendons) or caused by disease (e.g. distal diabetic osteomyelitis) are difficult to achieve therapeutic concentrations of antibiotics.
Passive diffusion across capillaries is the most common mechanism of tissue penetration but the blood-brain barrier has non-fenestrated capillaries reducing the ability of some antibiotics to pass. Lipophilic antibiotics such as metronidazole have an advantage in this situation over water-soluble antibiotics such as β-lactams, although increasing the dose of the latter can overcome these difficulties. Despite meningeal inflammation reducing the effectiveness of the blood-brain barrier, higher doses of β-lactams are used for suspected central nervous system (CNS) infections than for infections elsewhere.
Only the unbound (free) drug is active. The total serum concentration of a drug might be well above the concentration required to kill bacteria, however, if it is highly protein-bound, the unbound portion of the drug is likely to be less than the concentration required to kill bacteria.
Once in the infected tissues, there are several local factors that play a role:
Infected tissues tend to have a low pH which renders some antibiotics ineffective (e.g. gentamicin and erythromycin).
Local hypoxia. Penetration of the cell wall by aminoglycosides is by an oxygen-dependent reaction, therefore activity may be reduced in an anaerobic environment.
Presence of antimicrobial defences. Some bacteria produce substances that inactivate antibiotics (e.g. β-lactams).
Calcium ion concentration. This can adversely affect activity of some drugs (e.g. gentamicin).
Biofilm formation. A biofilm is a complex structure consisting of a variety of microorganisms and associated secreted substances. The resulting biofilm resists attack from host cells and antibiotics as well as adhering to foreign bodies.
After an intravenous bolus of drug, the serum concentration declines due to the initial distribution into tissues and then by elimination (metabolism and excretion). The elimination of antibiotics involves the hepatic and renal routes which can be to the clinician’s advantage but may also present a challenge in multi-organ failure.
β-lactams are excreted by glomerular filtration and tubular secretion. As described in earlier chapters, competition can occur between these secretion systems. Probenecid can be used to reduce excretion of penicillins, cephalosporins and some other β-lactams by competitively inhibiting the secretion of these substances via the active transport secretion mechanisms.
Gentamicin is concentrated in the urine by glomerular filtration. The concentration of gentamicin achieved in the urine may be high enough to treat an organism that is considered ‘resistant’ to concentrations achievable in other body tissues.
Erythromycin, azithromycin, moxifloxacin, clindamycin and rifampicin are mainly excreted by the liver into the bile.
Pharmacodynamics of Antibiotics
Appropriate treatment regimens are critical for antibiotic prescribing not just to maximise the success rate, but also to prevent the increasingly important issue of resistance.
Bacteriostatic antibiotics inhibit bacterial growth and subsequently allow host defences to kill the bacteria. Host defences are ineffective in states of generalised immunodeficiency or at certain anatomical sites in the early stages of certain infections e.g. the cerebrospinal fluid (CSF). Conversely bactericidal antibiotics actively kill the bacteria and are independent of host defences.
The minimum inhibitory concentration (MIC) is defined as the lowest concentration of antibiotic that prevents a defined number of bacteria in suspension becoming turbid after overnight incubation in vitro in standard conditions. It is therefore a measure of potency. Different samples of the same species will have different MICs (sensitive isolates will have low MICs and resistant isolates will have high MICs). The minimum bactericidal concentration (MBC) is the lowest concentration of antibiotic required to cause bacterial death. For bactericidal antibiotics, the MBC is similar to the MIC. For bacteriostatic drugs the MBC is many times greater than the MIC. The ‘cut-off’ MIC that decides whether a bacterial isolate is sensitive or resistant is called the ‘breakthrough MIC’. The MIC is measured after overnight aerobic incubation of a standard inoculum of bacteria in a low protein liquid medium at p. 7.2 at a standard temperature, and at a defined number of bacteria – all of which vary significantly from the actual site of infection.
It must be remembered that the MIC is an in vitro measurement and that the antibiotic concentration achieved in the various body cavities will vary widely. Areas with low antibiotic concentrations include abscess cavities and areas with poor blood flow. Areas with high concentrations include the bile and urine, due to active concentration of drug in these areas.
When an antibiotic is administered, the number of bacteria will decline due to bacterial death whilst the concentration of the unbound antibiotic is greater than the MBC. When antibiotic concentration falls below the MBC (but still are above the MIC) the bacterial count may remain stable or continue to decline as a result of killing by host defences.
Once the antibiotic concentration falls below the MIC, some suppression of bacterial growth can still occur. Several mechanisms are responsible for this:
The post antibiotic effect (PAE). The suppression of bacterial growth after short exposure to antibiotic even without a contribution from host defences.
The post antibiotic leukocyte enhancement (PALE). After exposure to an antibiotic, bacteria are more susceptible to attack by host defences.
Minimal antibacterial concentration (MAC). Antibiotic drug concentrations less than the MIC can alter morphology, slow rate of growth and prolong the PAE. Concentrations above the MAC but below the MIC cause this effect.
As antibiotic levels fall, eventually bacteria start to resume growth. This rate of growth depends on the activity of the host defences and the species of bacterium. Rapidly growing bacteria in immunocompromised individuals or at certain anatomical sites can double their numbers every 20 minutes. The next dose of antibiotic needs to be given before significant regrowth occurs, and there are three main pharmacodynamic classes.
1. Drugs with time-dependent bactericidal action. Slow bactericidal drugs that require levels to be above the MIC for as much time as possible. The magnitude of the concentration above the MIC has little relevance (i.e. more killing does not occur at higher concentrations). They have minimal post-antibiotic effect. These drugs need to be given frequently so that levels remain above the MIC as much as possible. Larger doses only work by increasing the duration of time the drug concentration is above the MIC. Giving multiple daily doses increases the time spent above the MIC. The drug concentration needs to be above the MIC for at least 50% of the dosing interval. Examples are penicillins and vancomycin. As a result, these drugs are very amenable to continuous infusions (following a loading dose) and this is currently being investigated although at the time of writing there is little evidence outcomes are better. For vancomycin and teicoplanin, in clinical practice, maintenance of trough serum levels of free drug that are greater than the MIC is most commonly recommended.
2. Drugs with a concentration-dependent bactericidal action. These drugs kill more bacteria the higher the concentrations they achieve (in contrast to above). They have significant and prolonged post-antibiotic effects. Efficacy depends on the maximum concentration achieved and the time spent above the MIC. The amount of drug rather than the frequency of dosing determines efficacy. Examples include aminoglycosides, fluoroquinolones, daptomycin, colistin, and metronidazole. Maximising serum concentrations of drugs that exhibit concentration-dependent bactericidal activity by increasing the dose will maximise the rate and extent of bactericidal activity, if adverse effects are not also concentration-dependent.
3. Drugs with predominately bacteriostatic actions. These have prolonged PAEs. Efficacy is determined by the time spent above MIC.
Antibiotic Combinations
Adding an aminoglycoside to a drug with a relatively slow rate of bactericidal activity (e.g. β-lactams or glycopeptides) can improve antimicrobial action. For example, a penicillin is slowly bactericidal, and an aminoglycoside alone at concentrations achieved in serum after standard dosing exhibits only bacteriostatic activity, but the combination of the penicillin with an aminoglycoside results in rapid bactericidal activity. The presence of a drug that affects cell wall synthesis (penicillin) enhances the penetration of the aminoglycoside.
However, this type of interaction between antibiotics can also be antagonistic. Penicillin is bactericidal and inhibits cell wall synthesis, weakening the wall and eventually causing cell lysis as it grows. Penicillin requires the organisms to be growing. Tetracycline prevents cell growth therefore reducing the susceptibility of some bacteria to penicillin.
1. Specific Antibiotics that Affect Cell Wall Synthesis β-lactams
β-lactam antibiotics are subdivided into the following classes:
Penicillins
Cephalosporins
Carbapenems
Monobactams.
Penicillins
Structure
Penicillins contain a β-lactam ring fused with a thiazolidine ring (see Figure 23.3). Structurally they differ in the type of acyl side-chain attached to the β-lactam ring, which determines both susceptibility to acid degradation in the stomach and spectrum of activity. Only penicillin V (acid resistant) and penicillin G (acid sensitive) are produced naturally, the others are semisynthetic (see Table 23.3). An intact β-lactam ring is essential for activity.
Table 23.3 Classification of penicillins
| Type | Examples | Indications |
|---|---|---|
| Narrow-spectrum, naturally occurring | benzylpenicillin (Penicillin G); phenoxymethylpenicillin (Penicillin V) | G+ve coccii (meningococcal infections) |
| Narrow-spectrum, semisynthetic | flucloxacillin | penicillinase producing staphylococcal infections |
| Narrow-spectrum, staphylococcal β-lactamase resistant | temocillin | G-ve lactamase-producinig bacteria (not pseudomonas) |
| Broad-spectrum penicillins | ampicillin, amoxycillin, co-amoxiclav | G+ve and some G-ve cover. Ampicillin and amoxicillin are increasingly less effective due to resistance. Co-amoxiclav has increased activity by combining amoxicillin with clavulanic acid, a β-lactamase inhibitor. |
| Antipseudomonal penicillins | piperacillin, ticarcillin | Broad specturm of activity against Gram+ve and G-ve bacteria and anerobes. |
Figure 23.3 Structures of penicillins and cephalosporins. R, variable groups on the β-lactam ring in penicillins; R1, R2, variable groups on the cepham ring structure.
Uses
Penicillins are used widely against a variety of infections and are most effective against Gram-positive bacteria.
Mechanism of Action
Penicillins are bactericidal antibiotics that inhibit cell wall synthesis by preventing peptidoglycan cross-linkage, resulting in weakening of the cell wall. The β-lactam ring resembles the natural substrate D-ala-D-ala on the side-chain of the peptidoglycan where cross-linkage occurs. Penicillins bind to several penicillin-binding proteins (PBP) in the cell wall that act as transpeptidase enzymes responsible for forming cross-links. On binding, the β-lactam structure is ruptured to form a bond with the active serine site on the PBP resulting in irreversible inactivation of the transpeptidase enzyme. Penicillin activity is lost in the presence of β-lactamase, which hydrolyses the β-lactam ring. β-lactamases are encoded on bacterial chromosomes and plasmids, which may be disseminated and lead to acquired resistance.
When Gram-positive bacteria are exposed to β-lactams, growth continues but with reduced cross-linkage of peptidoglycan until the cell wall becomes weakened and lysis is inevitable. The use of β-lactams against susceptible Gram-positive cocci rarely results in complete eradication of bacteria, a significant number of β-lactam sensitive cells, known as persistors, will remain dormant until the antibiotic is removed. Complete eradication can be achieved by the addition of a synergistic antibiotic, such as the aminoglycoside gentamicin, the potency of which is enhanced by cell-wall damage allowing better intracellular access.
The cell walls of Gram-negative bacilli are different and damage to the thin peptidoglycan layer only weakens it, since the thicker lipopolysaccharide outer layer remains intact. However, internal hydrostatic pressure forces the bacteria to become spherical (spheroplasts) which may lead to cell lysis. In some bacilli, such as Haemophilus influenzae, intracellular osmolality is so low that lysis rarely occurs. When the antibiotic is removed, peptidoglycan integrity is restored, their normal rod-like shape returns and they continue to divide normally.
Pharmacology
Penicillins are drugs with time-dependent bactericidal action. In order for bacteria to be killed the concentration of the drug must be above the MIC for as much time as possible.
Intestinal absorption depends on whether the type of penicillin is susceptible to acid-induced degradation in the stomach; amoxicillin is absorbed more readily than ampicillin. Some penicillins, such as benzylpenicillin (penicillin G), are so susceptible to acid hydrolysis that they are available only for parenteral administration. Plasma half-lives are short (benzylpenicillin, 30 minutes; ampicillin, 2 hours). Tissue penetration is generally good although inflammation is necessary for penicillins to pass into bone and through the blood–brain barrier. Penicillins are excreted by the kidneys unchanged (60–90%), mainly by renal tubular secretion, but they are also excreted in bile (10%) and up to 20% is metabolised.
Clavulanic acid and tazobactam are β-lactamase inhibitors. The combination of a penicillin with one of these agents can restore sensitivity in bacteria producing a β-lactamase enzyme. Amoxicillin is combined with clavulanic acid in co-amoxiclav and piperacillin with tazobactam. Clavulanic acid itself has weak antibacterial activity whereas tazobactam does not.
Side Effects and Special Points
Hypersensitivity – allergy to penicillins occurs in up to 10% of the population and anaphylaxis in 0.01% (1 in 10,000). Cross-reactivity exists between penicillins, cephalosporins and carbapenems and is discussed below.
Encephalopathy – benzylpenicillin is the most pro-convulsant β-lactam. Toxic CSF levels can be reached by intrathecal injection, using large intravenous doses in meningitis patients with renal impairment, and concomitant probenecid use.
Diarrhoea is common during oral therapy. Ampicillin is associated with a low risk of pseudomembranous colitis (0.3–0.7%).
Miscellaneous – ampicillin produces a maculopapular rash in 10% of all patients.
Cephalosporins
Structure
Cephalosporins possess a similar structure to penicillins but the β-lactam ring is fused with a dihydrothiazine ring (see Figure 23.3) to produce the cephem nucleus. The cephalosporins are less sensitive to β-lactamases and are extended-spectrum bactericidal antimicrobials classified by generation (see Table 23.4). With each generation Gram-positive cover is maintained but Gram-negative cover is steadily improved.
| Generation | Examples | Features |
|---|---|---|
| First | Cefaclor; cefalexin; cefradine | Narrow-spectrum, G+ve cocci |
| Second | Cefuroxime; cefaclor; cefoxitin | Narrow-spectrum, G+ve cocci; cefuroxime also effective against H. influenzae |
| Third | Cefotaxime; ceftazidime; ceftriaxone | Broader spectrum; enhanced resistance to β-lactamase; less potent against G+ve; Pseudomonas sensitive; cross BBB |
| (Fourth) | Cefepime; cefpirome | Active against Enterobacter and Pseudomonas |
| Fifth | Ceftaroline; ceftobripole | Also active against multi-drug resistant S. aureus (MRSA, VISA, VRSA) |
Uses
A wide range of Gram-positive infections.
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