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What are the structural and chemical characteristics that make tetracycline uniquely broad spectrum? I understand it acts on the A-site of the prokaryotic ribosome, but there exist many ribosome-targeted drugs that are relatively narrow spectrum. I assume it has something to do with its permeability in several different classes of bacterial cell walls and/or membranes - but I can't find any literature that links some characteristic part of its structure to this permeability.
This is a tricky question. First of all, I wouldn't call tetracyclines "uniquely" broad spectrum. They are broad spectrum, but there are other drugs with equally broad coverage absent acquired resistance (e.g., imipenem and chloramphenicol), and current clinical use of tetracyclines is limited somewhat by the spread of resistance. They don't have any major class limitations, though. Again, absent acquired resistance, tetracyclines are effective against gram positive aerobes and anaerobes, and gram negative organisms, as you seem to be aware.
Tetracylines access their target on the 30S subunit of the bacterial ribosome (blocking the A site, as you said) by passive diffusion through porins of the outer membrane of gram negative bacteria, and active transport across the cytoplasmic membrane of gram negative and gram positive bacteria, but the mechanism for that active transport is still unknown. This is quite a good review despite being quite old. Now, some 30 years later, we have still not identified the exact mechanism, as you can read in Goodman & Gillman's most recent edition (13th), Ch. 59, crossing the cytoplasmic membrane requires metabolic energy, but the process is not well understood. Because of this, we can't point to a specific structural or chemical characteristic. For chloramphenicol, it would be the ability to diffuse across the cytoplasmic membrane; for tetracycline, we don't know. I suspect part of the reason for this is that people are now more interested in pumps removing tetracyclines from the cell than those that accumulate it, so it's more of a question about research priorities than some transport mechanism that is particularly difficult to detect.
Tetracycline, sold under the brand name Sumycin among others, is an oral antibiotic in the tetracyclines family of medications, used to treat a number of infections,  including acne, cholera, brucellosis, plague, malaria, and syphilis. 
- 60-54-8 Y
- DB00759 Y
- 10257122 Y
- D00201 Y
- CHEBI:27902 Y
- ChEMBL1440 Y
Common side effects include vomiting, diarrhea, rash, and loss of appetite.  Other side effects include poor tooth development if used by children less than eight years of age, kidney problems, and sunburning easily.  Use during pregnancy may harm the baby.  It works by inhibiting protein synthesis in bacteria. 
Tetracycline was patented in 1953 and came into commercial use in 1978.  It is on the World Health Organization's List of Essential Medicines.  Tetracycline is available as a generic medication.  Tetracycline was originally made from bacteria of the Streptomyces type. 
TETRACYCLINE ANTIBIOTICS Full Text
Tetracycline antibiotics have a broad spectrum of activity, are relatively safe, can be used by many routes of administration, and are widely used. They even have antiprotozoal activity. The major difference among the tetracyclines is in their pharmacokinetic properties. Cross resistance among members of the group is frequent.
Structure and chemical characteristics
Four fused 6-membered rings, as shown in the accompanying figure, form the basic structure from which the various tetracyclines are made. The various derivatives are different at one or more of four sites on the rigid, planar ring structure. The classical tetracyclines were derived from Streptomyces spp ., but the newer derivatives are semisynthetic as is generally true for newer members of other drug groups. Stability of tetracyclines in solution varies with pH and derivative. The drugs are amphoteric, meaning they will form salts with both strong acids and bases. Thus, they may exist as salts of sodium or chloride.
There are no rigid subgroupings of the tetracyclines, but as you study this material, you might note how frequently their characteristics place them in one of the 3 classes below. These are based on dosage and frequency of oral administration. Group 1 includes such older derivatives as chlortetracycline (now little used), oxytetracycline , and tetracycline . Group 2 includes demeclocycline and methacycline . Group 3 includes newer drugs such as doxycycline and minocycline .
Mechanism of action
Tetracyclines bind reversibly to the small subunits of bacterial (and eukaryotic) ribosomes where they interfere with binding of charged-tRNA to the "Acceptor" site. They are "bacteriostatic" rather than cidal. Tetracyclines can also inhibit protein synthesis in the host, but are less likely to reach the concentration required because eukaryotic cells do not have a tetracycline uptake mechanism.
Tetracyclines are increasingly met by resistant organisms when used in clinical practice, but are still considered to be useful. Sensitive organisms accumulate tetracyclines intracellularly because of active transport systems. There are no known enzymes that inactivate the tetracyclines. Resistance to one tetracycline usually implies resistance to the others, although some research studies have observed differences in MICs for various tetracycline derivative - isolate pairs. These differences are not large and are not uniform throughout the country.
Resistance is transferred in plasmids that code for proteins that "pump" the drugs out of the cells. The intracellular concentration represents the balance between the input and output mechanisms. There is conceptual similarity in this resistance mechanism and that of cancer cells that develop resistance to different anticancer drugs in one step.
Note the similarity between doxycycline and minocycline pharmacokinetics in the discussion that follows. They are relatively new tetracyclines that have been developed to overcome deficiencies in the older derivatives.
Tetracyclines are primarily used by oral administration, but topical, IM, and IV forms exist. Only oxytetracycline and tetracycline have IM dose forms the others cause sterile abscesses. IV injections are given by infusion to avoid cardiovascular collapse. IV dose forms exist for minocycline, doxycycline, and the two that also have IM dose forms, oxytetracycline and tetracycline.
The tetracyclines vary widely in their bioavailability and the effect that food has on it. Doxycycline and minocycline have very high bioavailability, in the range of 90 to 100%, and the presence of food has an insignificant effect. The others have bioavailabilities approximating 58 to 77% and are significantly decreased by food. Calcium, aluminum, and magnesium form insoluble chelates with tetracyclines to decrease bioavailability. Milk is high in calcium and all of these ions are high in antacids so these should be avoided. Some laxatives have magnesium. Because tetracyclines are irritants that produce stomach upset, patients should be cautioned not to use milk or antacids to counteract the distress. Owners of animals should be similarly cautioned.
Doxycycline reaches therapeutic concentrations in the eye. Minocycline is also widely distributed, reaching high concentrations in saliva and tears. Both are used in treatment of genitourinary tract infections because they produce therapeutic concentrations in these tissues, including the prostate. All tetracyclines are distributed to most body fluids including such transcellular fluids as bile, sinus secretions, synovial, and pleural fluids. CSF concentrations are 10-25% of plasma concentrations. This is sufficiently low that they are not highly recommended for CNS infections.
Working with cattle, Ziv and Sulman (1974) found that approximately 20 minutes were required for intravenously administered doxycycline and minocycline to reach a milk:serum ratio over 1.5. Tetracycline and oxytetracycline took 60 minutes to reach ratios of 1.25 and 0.75, respectively. These values reflect the differences in ability of the drugs to cross membranes implied above. However, note that in all cases, the ratio approached 1 or more, a result not seen with such drugs as the beta-lactams or aminoglycosides.
Tetracyclines have high apparent volumes of distribution, ranging from 0.7 L/kg for doxycycline to as much as 1.9 for oxytetracycline. Tetracyclines typify the complexities of using Vd as an indicator of therapeutic concentrations in tissues. Tetracyclines localize in bones, teeth, liver, speen, and tumors. Because they are highly bound to these tissues and bone, they are non-homogeneously distributed outside the plasma. Paradoxically, doxycycline and minocycline cross membranes more easily than any of the others, but because high plasma protein binding offsets the accumulation in bone and other tissues, they have Vds of 0.14 to 0.7 L/kg.
Volumes of distribution for animals are in the same range as for humans, but significant differences do occur. For example, the Vd of minocycline is 1.9 L/kg in dogs versus 0.4 for humans. Oxytetracycline Vds are 1.4, 0.8, 2.1, and 2.1 L/kg for horses, cattle, dogs, and cats, respectively. Note that the value for humans, 0.9 to 1.9 L/kg, brackets the range for these species.
All tetracyclines are eliminated via renal and biliary pathways, but differ in their relative dependence on the two. All undergo significant enterohepatic circulation. Doxycycline and minocycline are primarily eliminated in the bile and less than a third is eliminated unchanged. Oxytetracycline, tetracycline, methacycline, and demeclocycline are eliminated primarily in urine with 42 to 70%, depending on the derivative, being eliminated unchanged.
Elimination half-lives range from 6-11 hours for tetracycline and oxytetracycline to 11 to 23 for doxycycline and minocycline. Anuria hardly changes the rate of elimination of doxycycline and minocycline, but tetracycline elimination half-life increases to 57 to 108 hours.
Elimination half-lives of oxytetracycline, tetracycline, and minocycline tend to be shorter in dogs, approximately 6 hours, than in humans (9.5, 10.6, and 17.5 hours, respectively). Horses and cattle have elimination half-lives of oxytetracycline similar to those of humans. [Horses/donkeys may have longer half-lives resulting in the toxicity frequently reported, Bowersock, T. 1995]
The data presented above for doxycycline and minocycline imply that they are biotransformed to some extent in the liver. Indeed, phenytoin or barbiturate induction of hepatic drug metabolizing enzymes may reduce the elimination half-life of doxycycline by more than 50%.
Tetracyclines are generally regarded as relatively non-toxic, but they produce a fairly large number of adverse effects, some of which can be life threatening under the right circumstances. Therefore, they should not be used casually.
Allergic reactions are not a major problem with the tetracyclines although they do occur.
Biological adverse effects
Superinfection (suprainfection) may occur with the tetracyclines, particularly the older, more poorly absorbed ones when given orally. Because of their broad spectrum of activity, activity against commensal organisms of the gut, and effective concentration in the gut, they nearly always alter the intestinal flora. This may occur within 24 to 48 hours, but these changes are not always clinically evident as diarrhea. It is not unusual to find superinfection with yeasts or resistant pathogenic bacteria. Although frowned upon by the FDA, commercial preparations of tetracyclines combined with nystatin (an oral antifungal) have been prepared to help combat superinfection with yeasts. Many authorities believe that because such superinfections do not always occur, there is less risk to the patient if one waits until there is evidence of yeast superinfection before beginning therapy.
Diarrhea may occur and will usually be the result of change in microflora of the gut. See the discussion of superinfection.
Indigestion may occur for reasons already presented under the heading of superinfections. It may be difficult to differentiate indigestion due to changes in flora from that caused by direct irritation to the gastrointestinal mucosa.
Indigestion is potentially problematic in ruminants because of the large number of bacteria and protozoans in the rumen. Horses, rabbits and other animals with large cecum/colon microfloral populations are also sensitive to the effects of tetracyclines.
Sore mouth and perineal itching
Sore mouth and perineal itching due to overgrowth of yeasts are "more frequent" according to the USPDI11th90.
Most direct toxicity is due to the irritant properties of the drugs, the inhibition of protein synthesis, or their predilection for bony tissues.
Irritation of gastric mucosa leading to cramps or burning of the stomach can be of such severity as to cause poor patient compliance. This often results in nausea and vomiting . Note that minocycline and doxycycline may be taken with food to reduce the impact of this irritation.
The same irritant properties also limit the use of these drugs for IM or SC injections where all cause pain and most cause sterile abscesses.
Deposition in calcified tissues
Deposition in calcified tissues, e.g., teeth can result in discoloration, especially when given during developmental stages. Higher doses given at inopportune stages of growth can result in bone deformation. Nearly everyone who received tetracyclines as a child will have teeth that fluoresce under a UV light source whether their teeth are stained brown or not.
Dizziness / light headedness is commonly seen with minocycline, but not the others. This is caused by vestibular or CNS toxicity and is of such severity and frequency that CDC has changed recommendations on its non-essential use.
Antianabolic effect resulting from decreased protein synthesis. In the presence of reduced renal function this is evident as azotemia and increased serum urea nitrogen (SUN).
photosensitivity may be associated with the use of all tetracyclines, but is especially a problem with demeclocycline. Patients should be kept out of heavy sunlight when receiving tetracyclines.
The list of diseases for which tetracyclines can be used is long, but because of increasing resistance it is becoming shorter. It is advised that the reader consult a "current therapy", a "medicine" textbook, or a reference such as USPDI to see the range and types of infections for which they are regarded as effective therapy. Because they are effective against a wide range of bacteria and many protozoans their applications are broader than many antibacterials.
Tetracyclines are effective in many infections caused by Gram-negative and Gram-positive bacteria. Examples include Brucella , Francisella , Pseudomonas pseudomallei , Neisseria gonorrhoea , and Treponema pallidum .
Many Pasteurellae and Borrelia hurgdorferi (Lyme disease) . Most common use in vet med is in combination with sulfas (e.g., sulfadimethoxine [Albon] with which they are synergistic. Used to treat most Strept, Staph, Pasteurella infections in cattle [Bowersock 1995].
In addition, tetracyclines are effective in Rickettsial infections, such as Q fever and Rocky Mountain Spotted Fever, as well as those caused by Mycoplasma and Chlamydia . The latter two are often causes of pneumonia and genitourinary tract infections. Psittacosis, caused by Chlamydia psittac i, is treated with tetracyclines.
Problematic cases of malaria and ameobiasis may benefit from tetracyclines given in conjunction with more specific anti-infective therapy.
Demeclocycline may also be used to treat a non-infectious problem known as syndrome of inappropriate (excess) antidiuretic hormone ( SIADH ). It acts by inhibiting ADH-induced water reabsorption in the kidney to induce water diuresis. It is apparent that when used as an anti-infectious agent, this diuresis may be considered as an adverse effect.
- Ziv & Sulman, Am. J. Vet. Res. 35:1197, 1974.
- USPDI, 11th edition, 1991
- USPDI, 15th edition, 1995
- BM6th88, Huber, W.G., Tetracyclines, in Veterinary Pharmacology and Therapeutics , 6th edition, eds. Booth, N.H. and McDonald, L.E., Iowa State University Press, 1988.
- Rang, H.P. and M.M. Dale. Pharmacology , Churchill Livingstone, New York 1987, Chapter 30.
- Bowersock, T., 1995. Personal communication.
1. What is the major basis for selecting one drug from among the tetracycline group? Assuming you answered pharmacokinetic properties, how could this be reconciled with the fact that specific tetracyclines are often recommended for specific infectious processes?
2. Minocycline used to be the recommended treatment for meningococcal carriers, but CDC in Atlanta no longer recommends this? What specific toxicity is associated with minocycline? What does the change in this recommendation imply about cost-benefit ratios for some uses of drugs?
3. In what way are the tetracyclines (and sulfonamides to be studied later) different from other antibacterials in their action on protozoans? Be able to name two protozoan diseases for which the tetracyclines are reasonable parts of the therapy.
4. Why do you suppose a group of drugs is generally regarded as non-toxic when they produce so many adverse effects?
5. You should be able to recognize and discuss the basis of each of the tetracycline adverse effects, e.g., superinfections, diarrhea, and increased SUN. You should be able to list and discuss at least two representative effects from each of the two important (for the tetracyclines) categories of adverse effects.
6. How does the cross resistance of bacteria to tetracyclines compare to that of the beta-lactams and aminoglycosides?
7. What special precautions must be taken with the tetracyclines when used P.O.? Which two are apparently not affected by this problem?
8. Why are many of the tetracyclines never used IM or SC?
9. Why is intravenous administration of tetracyclines dangerous, despite the fact that it is one of the important means of use? Note that some persons believe calcium chelation is the cause of this hypotension, but that is not necessarily true. Addition of calcium salts to infusions is not considered good practice. Slow administration is!
10. Explain how the apparent Vd of some tetracyclines can be greater than the total body water?
11. What is the effect of poor renal or hepatic function on the elimination rate of the tetracyclines. Name one that is primarily eliminated via the kidney and one that is primarily eliminated via the bile.
12. How could the concomitant administration of phenytoin or phenobarbital and a tetracycline like doxycycline result in a drug failure?
RESIDUES IN MEAT AND MEAT PRODUCTS | Feed and Drug Residues
S. Croubels , . D. Courtheyn , in Encyclopedia of Meat Sciences , 2004
The tetracyclines are a group of antibiotics originally derived from certain Streptomyces spp. The major representatives of the tetracycline group available for treating food-producing animals are tetracycline, oxytetracycline, chlortetracycline and doxycycline. The tetracyclines, which are broad-spectrum antibiotics, are used to treat, for example, respiratory disease in cattle, sheep, pigs and chickens, and they may be administered parenterally, orally or topically. After application of tetracyclines to animals, bound residues of the antibiotics can be found in the bones of slaughtered animals even months after treatment. These bound residues can possibly reach the food chain via contaminated meat (mechanically deboned meat) or meat and bone meal.
The chemical structure of tetracycline is shown in Figure 10 .
Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance
Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from each other using molecular methods including DNA-DNA hybridization with oligonucleotide probes and DNA sequencing. A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA. New tetracycline derivatives are being examined, although their role in treatment is not clear. Changing the use of tetracyclines in human and animal health as well as in food production is needed if we are to continue to use this class of broad-spectrum antimicrobials through the present century.
Structure of 6-deoxy-6-demethyltetracycline, the minimum…
Structure of 6-deoxy-6-demethyltetracycline, the minimum tetracycline pharmacophore.
Stereochemical and substitution requirements for…
Stereochemical and substitution requirements for optimum antibacterial activity within the tetracycline series.
As with other antibacterials, use of this drug may result in overgrowth of nonsusceptible organisms, including fungi. If superinfection occurs, discontinue antibacterial and institute appropriate therapy.
Treat all infections due to Group A beta-hemolytic streptococci for at least ten days.
Perform incision and drainage or other surgical procedures in conjunction with antibacterial therapy, when indicated.
Prescribing Tetracycline in the absence of proven or strongly suspected bacterial infection or a prophylactic indication is unlikely to provide benefit to the patient and increases the risk of the development of drug-resistant bacteria.
Information for Patients
Counsel patients that antibacterial drugs including Tetracycline should only be used to treat bacterial infections. They do not treat viral infections (e.g., the common cold). When Tetracycline is prescribed to treat a bacterial infection, tell patients that although it is common to feel better early in the course of therapy, the medication should be taken exactly as directed. Skipping doses or not completing the full course of therapy may (1) decrease the effectiveness of the immediate treatment and (2) increase the likelihood that bacteria will develop resistance and will not be treatable by Tetracycline or other antibacterial drugs in the future.
In sexually transmitted infections, when coexistent syphilis is suspected, perform dark field examinations before treatment is started and the blood serology repeated monthly for at least four months.
Since bacteriostatic drugs may interfere with the bactericidal action of penicillin, it is advisable to avoid giving Tetracycline in conjunction with penicillin or other bactericidal antibacterials.
Because the Tetracyclines have been shown to depress plasma prothrombin activity, patients who are on anticoagulant therapy may require downward adjustment of their anticoagulant dosage.
The concurrent use of Tetracycline and methoxyflurane has been reported to result in fatal renal toxicity.
Absorption of Tetracyclines is impaired by antacids containing aluminum, calcium or magnesium and preparations containing iron, zinc, or sodium bicarbonate.
Concurrent use of Tetracycline may render oral contraceptives less effective.
Carcinogenesis, Mutagenesis, Impairment of Fertility
Long-term animal studies are currently being conducted to determine whether Tetracycline hydrochloride has carcinogenic potential. Some related antibacterials (oxyTetracycline, minocycline) have shown evidence of oncogenic activity in rats.
In two in vitro mammalian cell assay systems (L 51784y mouse lymphoma and Chinese hamster lung cells), there was evidence of mutagenicity with Tetracycline hydrochloride.
Tetracycline hydrochloride had no effect on fertility when administered in the diet to male and female rats at a daily intake of approximately 400 mg/kg/day, roughly 8 times the highest recommended human dose based on body surface area.
Pregnant women with renal disease may be more prone to develop Tetracycline-associated liver failure.
Labor and Delivery
The effect of Tetracyclines on labor and delivery is unknown.
Because of potential for serious adverse reaction in nursing infants from Tetracyclines, a decision should be made whether to discontinue the drug, taking into account the importance of the drug to the mother (see WARNINGS ).
Mode of Action:
The antimicrobial activity of tetracyclines reflects reversible binding to the bacterial 30S ribosomal subunit, and specifically at the aminoacyl-tRNA acceptor ("A") site on the mRNA ribosomal complex, thus preventing ribosomal translation. This effect also is evident in mammalian cells, although microbial cells are selectively more susceptible because of the greater concentrations seen. Tetracyclines enter microorganisms in part by diffusion and in part by an energy-dependent, carrier-mediated system responsible for the high concentrations achieved in susceptible bacteria. The tetracyclines are generally bacteriostatic, and a responsive host-defense system is essential for their successful use. At high concentrations, as may be attained in urine, they become bactericidal because the organisms seem to lose the functional integrity of the cytoplasmic membrane. Tetracyclines are more effective against multiplying microorganisms and tend to be more active at a pH of 6–6.5. Antibacterial efficacy is described as time dependent.
The most common mechanism by which microbes become resistant to tetracyclines is decreased accumulation of drug into previously susceptible organisms. Two mechanisms include 1) impaired uptake into bacteria, which occurs in mutant strains that do not have the necessary transport system, and 2) the much more common plasmid- or transposon-mediated acquisition of active efflux pumps. The genomes for these capabilities may be transferred either by transduction (as in Staphylococcus aureus) or by conjugation (as in many enterobacteria). A second mechanism of resistance is the production of a "protective" protein that acts by either preventing binding, dislodging the bound drug, or altering the negative impact of binding on ribosomal function. Among the tetracyclines, tigecycline is characterized by less resistance due to efflux or ribosomal protection. Rarely, tetracyclines can be destroyed by acetylation. Resistance develops slowly in a multistep fashion but is widespread because of the extensive use of low concentrations of tetracyclines.
All tetracyclines are about equally active and typically have about the same broad spectrum, which comprises both aerobic and anaerobic gram-positive and gram-negative bacteria, mycoplasmas, rickettsiae, chlamydiae, and even some protozoa (amebae). Tetracyclines generally are the drug of choice to treat rickettsiae and mycoplasma. Among the susceptible organisms is Wolbachia, a rickettsial-like intracellular endosymbiont of nematodes, including Dirofilaria immitis. Strains of Pseudomonas aeruginosa, Proteus, Serratia, Klebsiella, and Trueperella spp frequently are resistant, as are many pathogenic Escherichia coli isolates. Even though there is general cross-resistance among tetracyclines, doxycycline and minocycline usually are more effective against staphylococci.
Outlook For The Future
The link between the use of antibiotics and development of bacterial resistance has been established during the past 60 years. How quickly a particular pathogen acquires tetracycline resistance depends on a number of factors, many of which remain poorly defined. A few new tetracycline compounds are being examined or are in clinical trials, but it is unlikely that many more derivatives will be available in the near future. Unfortunately, the bacteria do not distinguish use for treatment of bacterial infection from use in treatment of noninfectious conditions. This is a concern, because the use of tetracycline is much broader than it was 20–30 years ago. The global consumption of tetracycline per year is not known. One can hypothesize that tetracyclines will be used increasingly against protozoan parasitic diseases and perhaps other parasitic diseases in the future. The role of antibiotics in food production and the impact of this on bacterial resistance of human pathogens has been a topic of considerable interest, and a recent supplement of Clinical Infectious Diseases [ 24] goes into great detail on the subject. Perhaps of greatest concern is the long-term subtherapeutic use of tetracyclines for treatment of nonbacterial noninfectious conditions. This type of low-level, long-term use puts significant selective pressure on the bacteria carried by the host and in the environment of the host being treated. If nothing is done, the usefulness of tetracycline as an antibacterial agent is limited as bacterial resistance increases. Therefore, we need to reduce all types of use of this agent and of all antibiotics being used worldwide if we hope to keep tetracycline therapy as an option for this century.
Finally, biological terrorism has become a real threat during the past year. It has been suggested that the most likely bacterial weapons would be Bacillus anthracis, Francisella tularensis, and/or Yersinia pestis [ 11]. In each case, doxycycline is important for treatment and/or prophylaxis ( table 1). Unfortunately, tetracycline-resistant Y. pestis has already been described, but it is rare [ 1]. However, last year, during the anthrax attacks, the general public accumulated antibiotics at home and often took these antibiotics in anticipation of exposure without doctor's consultation [ 11]. This unnecessary exposure to antibiotics by large numbers of people may lead to increased prevalence of antibiotic-resistant bacteria in the community, both for the 3 agents that could be used in biological weapons and for any other pathogenic bacteria. Thus, education of the public, health authorities, and clinicians is needed now more than ever to eliminate home stockpiling of antibiotics, to ensure the correct use for all bacterial infections (especially during a biological attack), and to prevent use when antibiotics are not needed, such as use for treatment of viral infections.
Tetracycline antibiotics used clinically include doxycycline, minocycline, and tetracycline, while tigecycline, which has the same four-ringed structure and is a derivative of minocycline, is the first member of the glycylcyclines to be approved their structures are shown in Figure 4.3.1 and their therapeutic indications are listed in Table 4.3.1.
Table 4.3.1 Therapeutic indications for the tetracycline antibiotics.
|Doxycycline||Chronic prostatitis, sinusitis, syphilis, uncomplicated genital chlamydial infection, pelvic inflammatory disease, acne vulgaris, rosacea, Lyme disease, community-acquired pneumonia|
|Minocycline||Acne vulgaris, prophylaxis of asymptomatic meningococcal carrier state (no longer recommended)|
|Oxytetracycline||Acne vulgaris, rosacea|
|Tetracycline||Acne vulgaris, rosacea, non-gonococcal urethritis, chronic bronchitis|
|Tigecycline||Complicated intra-abdominal or skin/soft tissue infections|
The tetracycline antibiotics are the third example so far in this section, of naturally occurring molecules from a microbial source that interfere with bacterial protein synthesis. Once again, the first in this series, chlortetracycline (originally named aureomycin), resulted from a programme of screening soil microorganisms for potential new antibiotics. This discovery is attributed to Benjamin M. Duggar, a retired botanist, with expertise across a wide range of plant and microorganism physiology. He retired from Wisconsin University in 1943, when he was 71, but was approached to act as consultant for Lederle Laboratories in New York (part of American Cyanamid Company, now part of Wyeth Pharmaceuticals), who were supporting the war effort by searching for new antibiotic and antimalarial agents (Walker, 1982).
Although he is solely credited with the discovery of chlortetracycline in 1948, Duggar was part of a larger team, under the direction of Yellapragada SubbaRow, which was systematically investigating soil microorganisms for natural products with desirable pharmaceutical activities. Duggar requested some local samples from the soil microbiologist at the University of Missouri, William Albrecht, from which he cultured a golden mould, which produced a yellow pigment that displayed growth inhibitory properties against bacteria, such as streptococci. He identified the mould as a Streptomyces species that had not previously been catalogued to reflect its colour, he named it Streptomyces aureofaciens and the antibiotic it produced aureomycin (Duggar, 1948). Note that the ‘mycin’ part of the name corresponds with its isolation from a Streptomyces species, like the aminoglycoside streptomycin and the macrolide erythromycin, which we have just met.
Aureomycin was quickly released to clinicians and other researchers to obtain evaluative data of its activity and efficacy it gathered support with glowing testimonials of its broad spectrum of activity, including against streptomycin- and penicillin-resistant organisms (Wright and Schreiber, 1949 Cantor, 1950 Kiser et al ., 1952). It was found to be as effective as penicillin and streptomycin and had the significant advantage of being the first antibiotic that was effective when administered orally.
Following the discovery of aureomycin, other tetracyclines were soon discovered:
- Oxytetracycline (originally called terramycin) in 1949 from Streptomyces rimosus by Pfizer (Finlay et al ., 1950).
- Tetracycline (marketed originally as achromycin (Darken et al ., 1960)) in 1953 from S. aureofaciens when cultured with a chlorination inhibitor (Goodman and Matrishin, 1968).
- Demethylchlortetracycline in 1957 from S. aureofaciens (McCormick et al ., 1957 Wilson, 1961). This was the last natural tetracycline to be identified and was originally called declomycin or ledermycin. It is still marketed as the latter (or under its generic name, demeclocycline) by Lederle Laboratories.
The discovery of this new class of antibiotics is not without controversy, though: Pfizer, American Cyanamid, and Bristol-Myers formed a monopoly that maintained artificially high prices for tetracycline over several years before the US Federal Trade Commission halted the violations after a series of high-profile investigations, charges, and appeals heard in the high court (Anon, 1964 US Court of Appeals, 1968).
When it was discovered that the hydrogenation of chlortetracycline resulted in dechlorination and conversion into tetracycline (which was as active as chlortetracycline) (Stephens et al ., 1952 Conover, 1955), the possibility that synthetic modification of tetracyclines might provide alternative agents with antibacterial activity was realised. During the next 15–20 years, many semi-synthetic analogues were prepared, including lymecycline, doxycycline, and minocycline some of these second-generation semi-synthetic tetracyclines were even more potent than chlortetracycline and are still marketed today. Structural modification has continued and has resulted in the discovery of a third-generation tetracycline, t -butylglycylamidominocycline (tigecycline, originally labelled GAR-936) (Petersen et al ., 1999), with more in development and in clinical trials (Sun et al ., 2008 Brötz-Oesterhelt and Sass, 2010).
Structurally, the tetracyclines are based on a four-ring (tetracyclic or octahydronaphthacene) system, hence the name the rings are labelled A, B, C, and D (Figure 4.3.2). One face consists of carbonyl, phenol, alcohol, and enol oxygen atoms, with high polarity and metal ion binding ability, while the other face is substantially less polar. There are a number of substitution patterns commonly found in the antibiotic tetracyclines and significant deviation from these leads to greatly reduced antibacterial activity (Chopra and Roberts, 2001 Zhanel et al ., 2004).
Figure 4.3.2 Requirements for tetracycline antibiotic activity (Chopra and Roberts, 2001 Zhanel et al ., 2004)
Much of the development of new tetracycline antibiotics has been driven by the instability of the first-generation tetracyclines, particularly chlortetracycline, oxytetracycline, and tetracycline, which can lead to degradation during storage and even production of a toxic product. We will look at some of the reactions of tetracyclines and the resultant effects upon bioavailability in Subsection 4.3.3.
Looking back at Figure 4.3.1, you can see that the tetracyclines have a number of chiral centres and functional groups, so you will not be surprised to learn that fermentation methods are considered to be the most cost-effective for their production, and for the production of the base structures for semi-synthetic analogues, such as lymecycline and tigecycline (Khosla and Tang, 2005). As you will see further on in this subsection, there is now an efficient chemical synthetic method for multigram quantities of a key intermediate in tetracycline synthesis, which offers the possibility of analogue synthesis (Brubaker and Myers, 2007). The first patented fermentations of S. aureofaciens were for the production of chlortetracycline (Duggar, 1948 Neidercorn, 1952) and tetracycline (Goodman et al ., 1959) much work since then has focussed on optimising the selectivity for, and the yields of, the desired tetracyclines, especially since some Streptomyces species can produce more than one tetracycline, depending upon the fermentation conditions (Bêhal, 1987, 2000). In early 2011, there were almost 3000 patents relating to the biosynthesis and synthesis of tetracycline and its analogues ( worldwide.espacenet.com ), including some filed in 2010 and 2011 – proof that there is still interest in the production and use of tetracyclines. It should be noted, however, that some of these were for non-antibiotic uses of tetracyclines, briefly mentioned in Subsection 4.3.4.
188.8.131.52 Biosynthesis of Tetracyclines
The biosynthesis of tetracycline antibiotics is related to the bacterial synthesis of fatty acids through the bacterial type II polyketide synthase pathway, consisting of a well-studied set of enzymes (for examples, see Khosla, 2009 Zhang and Tang, 2009), although the synthesis of tetracyclines is unique to certain bacteria (Clardy et al ., 2009). The biosynthetic pathways to tetracycline and oxytetracycline are the most studied (for example, Petkovi et al ., 2006 Pickens and Tang, 2009). The biosynthetic pathway to natural tetracyclines is available from the KEGG database ( www.genome.jp/kegg/pathway/map/map00253.html , last accessed 10 March 2012) in summary, it uses the precursor, malonamyl coenzyme A (CoA), which is obtained from acetyl CoA via malonyl CoA and glutamine (Wang et al ., 1986), and proceeds through two common key intermediates, 6-methylpretetramide and 4-ketoanhydrotetracycline (Scheme 4.3.1 and Table 4.3.2) (Clardy et al ., 2009 Pickens and Tang, 2009).
Scheme 4.3.1 Biosynthetic pathway to tetracycline antibiotics (Clardy et al ., 2009 www.genome.jp/kegg/pathway/map/map00253.html , last accessed 10 March 2012)
Table 4.3.2 Enzymes involved in tetracycline biosynthesis.
|OxyB||Chain length factor|
|OxyC||Acyl carrier protein|
|OxyS||Monooxygenase that hydroxylates stereospecifically at C6|
For clarity, in Scheme 4.3.1 the precursor unit, malonamyl CoA, is coloured pink throughout the consecutive acetyl units added to malonamyl CoA from acetyl CoA are coloured alternately black and red – you can see that the cycle of acetyl addition occurs eight times until the linear nonaketamide is formed. A series of enzymic reactions involving OxyJ, OxyK, and OxyN leads to pretetramide (not shown in Scheme 4.3.1), which is converted into 6-methylpretetramide by OxyF. This series of reactions results in cyclisation of the nonaketamide to the tetracyclic structure of 6-methylpretetramide, followed by methylation at C6, with the new bonds formed in this sequence shown in blue. If you trace the sequentially added acetyl units, you can see how the two-carbon units form the backbone of the structure. Oxidation at C4 (by OxyE), and hydroxylation at C12a by OxyL, provides the second key intermediate, 4-ketoanhydrotetracycline, at which the tetracycline biosynthetic paths diverge. Chlortetracycline results from Cts4 halogenase action at C7 (Dairi et al ., 1995), followed by amination at C4 (OxyQ) and OxyT N , N -dimethylation (the methyl groups are provided by S -adenosyl methionine) OxyS catalyses the stereospecific hydroxylation at C6 , leaving a stereospecific reduction at C5a required to produce chlortetracycline. In the other pathway, amination at C4 , dimethylation , and hydroxylation at C6 provide 5a,11a-dehydrotetracycline, from which oxytetracycline and tetracycline are obtained.
Much detailed research has been directed at elucidating this pathway most was carried out on the enzymes of the oxytetracycline-producing species Streptomyces rimosus , hence the Oxy names, but the enzymes are the same or similar for the other tetracycline-producing species (Zhang et al ., 2007 Petkovi et al ., 2010 Pickens and Tang, 2010).
184.108.40.206 Chemical Synthesis of Tetracyclines
The literature related to the chemical synthesis of tetracyclines resembles a ‘Who’s Who’ of synthetic organic chemistry:
- R. B. Woodward solved the structure, complete with stereochemistry, in 1952 (this was revised slightly in the 1960s with the help of X-ray crystallography) (Hochstein et al ., 1953 Donohoe et al ., 1963 von Wittenau et al ., 1965).
- Woodward and Conover (who first synthesised tetracycline by hydrogenation of chlortetracycline) synthesised a biologically active tetracycline, named sancycline (Korst et al ., 1968), albeit in 25 steps and 0.002% overall yield.
- Shemyakin synthesised a tetracycline natural product, (±)-12a-deoxy-5a,6-anhydrotetracycline (Gurevich et al ., 1967).
- Muxfeldt identified the major problems with the synthesis of tetracyclines: the complexity of the required stereochemistry and the sensitivity of the tetracycline functional groups to both mild acid and base during initial studies (Muxfeldt and Rogalski, 1965), then later achieved the total synthesis of (±)-5-oxytetracycline in 22 steps and 0.06% (Muxfeldt et al ., 1968, 1979).
- Stork concentrated on achieving the correct stereochemistry at each centre in the basic tetracycline structure, producing (±)-12a-deoxytetracycline in 16 steps and an impressive 18–25% yield, although this structure has little antimicrobial activity (Stork et al ., 1996).
- Tatsuta and co-workers exploited the natural stereochemical definition of carbohydrates for their starting materials and achieved the total synthesis of natural (−)-tetracycline from D-glucosamine in 34 steps and 0.002% yield (Tatsuta et al ., 2000 Tatsuka and Hosokawa, 2005), including a solution to the difficult stereospecific hydroxylation of C12a.
- More recently, Myers and co-workers developed a highly effective synthetic approach to natural tetracyclines, their analogues, and their precursors (Charest et al ., 2005a, 2005b Brubaker and Myers, 2007 Myers et al ., 2007, 2011 Sun et al ., 2008), which takes account of the considerable challenge in achieving the correct stereochemistry, particularly at C12a, and provides versatility for the synthesis of many new analogues for microbiological evaluation (Myers et al ., 2007 Sun et al ., 2008).
A discussion of the chemical strategies for synthesising tetracyclines, the reactions required, and their stereochemical complexities would be a section in itself, so we will restrict ourselves here to consideration of the most recent syntheses, which have enabled multigram quantities of optically pure tetracyclines to be achieved and thus offer potential commercial synthetic routes to these agents. Myers and colleagues recognised that one key intermediate 1 , providing the A and B rings of tetracyclines, allows the synthesis of a wide range of tetracycline antibiotics and their analogues (Scheme 4.3.2).
Scheme 4.3.2 Key intermediates in the synthesis of tetracycline antibiotics (Myers et al ., 2007, 2011)
Initially, they developed a synthesis to this important intermediate from benzoic acid and achieved intermediate 1 in 21% overall yield after 7 steps (Charest et al ., 2005a). Using this route, (−)-tetracycline could be synthesised in 17 steps and 1.1% yield from benzoic acid. Conversion of 1 into 2 and 3 provided routes to tetracyclines with no hydroxyl group at C5 (including an alternative synthesis of tetracycline) and 5-hydroxytetracyclines, respectively. Using this approach, the synthesis of (−)-6-deoxytetracycline was achieved in 14 steps and 7% yield via intermediate 2 , while (−)-doxycycline was isolated in 8.3% yield after 18 steps via intermediate 3 the yields and number of steps relate to the total synthesis from benzoic acid (Charest et al ., 2005a, 2005b Myers et al ., 2007, 2011). Not content with these impressive achievements, Myers and Brubaker re-designed and improved the synthesis of intermediate 2 (Scheme 4.3.3), from which most tetracyclines can be accessed they identified an alternative cheap and readily available commercial starting material, methyl 3-hydroxy-5-isoxazolecarboxylate 4 , along with an improved synthetic strategy to improve stereoselectivity and yields, and obtained intermediate 2 in 9 steps and 21% overall yield from starting material 4 (Brubaker and Myers, 2007).
Scheme 4.3.3 Improved synthesis of key intermediate 2 for large-scale tetracycline synthesis (Brubaker and Myers, 2007)
There are several noteworthy features in this revised synthesis of intermediate 2 (Scheme 4.3.3):
- First, the introduction of a stereogenic (chiral) centre into structure 5 is carried out in high yield and with a high enantiomeric excess, and this enantiomeric ratio is maintained during the S N 2 replacement of the hydroxyl group (as a mesylate) with a dimethylamino group in the synthesis of 6 .
- The resulting stereogenic centre at C6 becomes C4 in the tetracycline and already has the correct stereochemistry, which is retained throughout the remainder of the synthesis.
- Intermediate 7 has a new stereogenic centre bearing a hydroxyl group you may be concerned that the stereochemistry is not defined at this new centre, but this group is oxidised to a carbonyl during the synthetic sequence that results in intermediate 8 , so the mixed stereochemistry does not matter at this stage.
So far, we have not considered how rings C and D can be constructed, yet this is just as important for the tetracycline structure. The synthetic strategy adopted by the Myers group uses intermediate 1 , 2 , or 3 as appropriate to provide rings A and B of the tetracycline with the correct stereochemistry and functionality, then elaborates this basic structure by construction of the C-ring, while adding the D-ring through a generalised Michael–Dieckmann reaction sequence (using a carbanion formed from a variety of D-ring precursors), followed by deprotection of all the functional groups (Scheme 4.3.4) (Charest et al ., 2005a).
Scheme 4.3.4 Elaboration of intermediates 2 and 3 into tetracyclines and their analogues (Charest et al ., 2005a)
One great advantage of this approach is the ease with which varying functionality can be added throughout the structure, particularly substituents at C5 ( X ), C6 ( R ), C7 ( Y ), C9 ( Z ), and even an extra ( E ) ring a great many analogues have been made, and some of these combine strong antibacterial activity with activity against strains resistant to first- and second-generation tetracycline antibiotics (Myers et al ., 2011 Sun et al ., 2011). The synthetic routes to the tetracyclines developed by the Myers group have been sufficiently successful to support a spin-out company, Tetraphase, which has several tetracyclines in early clinical trials. Other groups have also pursued C9-substituted tetracyclines (for example, Koza and Nsiah, 2002 Sum et al ., 2006) the clinical success of tigecycline (see Subsections 4.3.4 and 4.3.5) and the development of amadacycline (which is in clinical trials and is discussed in Subsection 4.3.9) provided the rationale for their evaluation.
The pharmacokinetics and pharmacodynamics of the tetracycline antibiotics were reviewed recently (Agwuh and MacGowan, 2006 Barbour et al ., 2010), but they are not fully understood, with several seemingly contradictory observations. The tetracyclines display time-dependent effects, yet the general concentration-dependent parameters (of exposure time at a concentration above the MIC) provide good clinical results, with a strong post-antibiotic effect. Although generally considered to be bacteriostatic, there is evidence of bactericidal activity with certain tetracycline antibiotics against specific bacteria, when used at an appropriate concentration (Zhanel et al ., 2004 Barbour et al ., 2010).
The instability of the first-generation tetracyclines during storage has already been mentioned we will consider the reactions of tetracyclines more carefully here, as they have an effect upon bioavailability and even upon the safety of the products.
The first-generation tetracyclines, although clinically successful, were found to be unstable to acidic, basic, and neutral pH during storage and in solution, including in the gastrointestinal (GI) tract after administration, decreasing their bioavailability (Walton et al ., 1970 Ali and Strittmatter, 1978 Wu and Fassihi, 2005). Two main reactions occur in the presence of acid: epimerisation at C4, to produce epitetracycline (Hussar et al ., 1968) (Scheme 4.3.5), and dehydration of 6-hydroxytetracyclines across C5a-C6 (with loss of the OH group at C6), to give the anhydrotetracycline derivative, as demonstrated for tetracycline in Scheme 4.3.6.
Scheme 4.3.5 Acid-catalysed epimerisation at C4 of tetracycline
Scheme 4.3.6 Acid-catalysed dehydration of chlortetracycline and improved stability of demethylchlortetracycline (demeclocycline)
The dehydration (shown in the upper part of Scheme 4.3.6 for chlortetracycline) proceeds through an E1 mechanism, involving protonation of the C6-OH and loss of a good leaving group (H 2 O), to produce a stabilised tertiary carbocation at C6 , followed by loss of a proton from C5a to form the alkene group of anhydrotetracycline. The slightly improved acid stability of demeclocycline and slower dehydration is due to fact that the secondary carbocation that has to be formed at C6 is less stable, and this process thus has a greater activation energy. In this latter case, reversible protonation of the C6-OH favours demeclocycline, instead of proceeding to the carbocation (Scheme 4.3.6).
Epimerisation at C4 of the anhydrotetracycline derivatives can also occur, producing the corresponding epianhydrotetracycline derivative (Sokoloski et al ., 1977). Some of the products formed by acid- or base-catalysed degradation are themselves active as antibiotics, while others, for example anhydrotetracycline and epianhydrotetracycline, are toxic (Mull, 1966 Kunin, 1967). The adverse effects (which can manifest as Fanconi syndrome see Subsection 4.3.7) of using tetracyclines that have degraded during storage were observed soon after these agents had been adopted into regular use, but it was several more years before reliable analytical methods for their analysis and quality control were developed (Frimpter et al ., 1963 Gross, 1963 Butterfield et al ., 1973). We will discuss the adverse effects of tetracyclines in Subsection 4.3.7 our main concern here is that the bioavailability of some tetracyclines (particularly the first-generation members) can be reduced by their degradation during storage, particularly in solution (Wu and Fassihi, 2005), or as a result of GI-induced reactions (Okeke and Lamikanra, 1995 Dos Santos et al ., 1998). The second-generation tetracyclines, doxycycline and minocycline, and the third-generation, tigecycline, are more stable to acidic pH, as they do not have a C6-OH substituent to be protonated and eliminated as water.
Lymecycline is a prodrug form of tetracycline that is hydrolysed at acidic and neutral pH in vivo to tetracycline, formaldehyde (methanal), and the amino acid lysine after oral and parenteral administration (Scheme 4.3.7). Interestingly, it has lower oral bioavailability than the parent compound, tetracycline (Sjölin-Forsberg and Hermansson, 1984).
Scheme 4.3.7 In vivo hydrolysis of lymecycline to form tetracycline
The balance of lipophilicity to hydrophilicity in tetracyclines is affected by pH (Chen and Lin, 1998) such a property is usually a clue that there are functional groups in the molecule under investigation which are ionisable at physiological pH values. Each tetracycline has at least one readily ionisable amine group (at C4) and two enol groups (at C3 and C12) that are also relatively easily ionised. First- and second-generation tetracyclines have three pK a values in the physiological pH range (generally around 3.2, 7.6, and 9.6) – the two enol groups, at C3 and C12 , are the most acidic (and so have the lower pK a values). The pK a of the phenol group at C10 (
12) means that it is not ionised at physiological pH values. The acid–base equilibria for tetracycline, which are typical of the tetracycline antibiotics, are demonstrated in Scheme 4.3.8 (Jin et al ., 2007).
Scheme 4.3.8 Acid–base equilibria for tetracycline (Jin et al ., 2007)
The fully protonated form of the tetracyclines (with an overall charge of +1) is likely to predominate in the acidic medium of the stomach, minimising absorption from this compartment (as neutral species are absorbed best). It is not surprising to find that tetracycline antibiotics are absorbed chiefly from the duodenum, where the pH is around 6–6.5 and the overall neutral form of the tetracycline predominates (Colaizzi and Klink, 1969). By modifying the pH, greater aqueous solubility of the tetracycline antibiotics can be achieved, making them suitable for parenteral administration, and oxytetracycline, lymecycline, doxycycline, and minocycline have been formulated in this way (Chopra and Roberts, 2001).
The third-generation antibiotic tigecycline has limited oral bioavailability and is administered by IV infusion, due to its greater hydrophilicity and reduced lipophilicity as a result of its extra ionisable groups (Meagher et al ., 2005). Tigecycline has two extra ionisable groups (Figure 4.3.3):
Figure 4.3.3 Tigecycline pKa values (Tygacil_BioPharmr)
- The t -butylamine on the side chain at C9 (a secondary aliphatic amine, so expected to be basic, with a pK a (of the conjugate acid, R 3 NH + ) of 8.5–10).
- The dimethylamino group at C7 (an aromatic amine, so expected to be a weak base, due to resonance of the nitrogen lone pair with the π-system of the aromatic ring, with a pK a of 3.5–5).
Besides being affected by pH, the absorption of the first-generation tetracyclines in particular, and some of the second-generation analogues, is adversely affected by their concurrent administration with food (Schimdt and Dalhoff, 2002 Agwuh and MacGowan, 2006), dairy products, and other metal-ion-containing preparations (such as antacid treatments), although there is some evidence that the absorption of lymecycline is less affected by milk (Ericson and Gnarpe, 1979). Tetracycline has a logP value of 0.09 and a bioavailability of around 75%, which is reduced by about 50% when co-administered with food (Miller et al ., 1977 Zhanel et al ., 2004). By comparison, doxycycline and minocycline have a greater bioavailability (90–100%) (Zhanel et al ., 2004), their absorption is not affected by food, and they are well absorbed after oral administration. The greater lipophilicity of these agents (logP values of 0.95 and 1.12, respectively) (Colaizzi and Klink, 1969) undoubtedly make a major contribution to these properties. Although minocycline offers improved oral bioavailability over the majority of other tetracyclines, the risk of side effects when used for a protracted period and of the development of resistance has limited it largely to the treatment of acne vulgaris. A recent review suggests that it may offer potential for the systemic treatment of community-associated MRSA and of the significant nosocomial threat of Acinetobacter baumanii (Bishburg and Bishburg, 2009).
Tetracyclines are known to bind strongly to a range of metal ions, with the strongest and most significant binding, in terms of bioavailability, mode of action, mechanisms of resistance, and adverse events, being to magnesium, calcium, iron, and copper (Agwuh and MacGowan, 2006). Chelation of tetracyclines to metal ions in the GI tract adversely affects the absorption of both the tetracycline and the metal ion through the precipitation of insoluble metal-tetracycline complexes (Agwuh and MacGowan, 2006), which provides a scientific rationale for avoiding the administration of tetracyclines alongside metal ion preparations and dairy products. The ability of tetracyclines to coordinate metal ions means that these antibiotics should not be administered to children, due to their ability to sequester calcium and other metal ions at a crucial time in bone and teeth development. In the USA and Australia, children under the age of eight are contraindicated, while in the UK tetracyclines should not be given to children under the age of 12. European guidelines on the third-generation tetracycline tigecycline recommend that it is not used for children and adolescents under the age of 18 years, due to lack of data on its safety and efficacy in these patient groups. The reason for the strong metal ion binding can be seen by considering the tetracycline structure – the lower face of the molecule (as can be seen in Scheme 4.3.9) has several oxygen atoms and so is ideal for binding to a metal ion the C12 (as the enolate) and C11 oxygen atoms are accepted to be the major binding site (Jin et al ., 2007 Palm et al ., 2008).
Scheme 4.3.9 Tetracycline-magnesium ion chelation (Jin et al ., 2007 Palm et al ., 2008)
We will return to tetracycline-magnesium complexes again later in this section, when we consider the uptake of tetracyclines into bacterial cells and also when we look at the mode of action (Subsection 4.3.4) and mechanisms of resistance (Subsection 4.3.5).
In general, the tetracycline antibiotics are not metabolised, except for tetracycline, of which about 5% is metabolised, and tigecycline, of which 5–20% is metabolised (Meagher et al ., 2005) they are excreted by both the urinary (<50%) and the faecal (>40%) routes (Agwuh and MacGowan, 2006). The urinary excretion of tetracyclines has been found to be affected by pH as expected, it is significantly increased at pH values above 8, at which values greater ionisation and hydrophilicity are also to be expected (Jaffe et al ., 1973).
The tetracyclines exhibit a high volume of distribution and generally good tissue penetration alongside their long half-lives and significant post-antibiotic effect, most can be administered once or twice daily (Table 4.3.3).
Table 4.3.3 Pharmacokinetic parameters for selected tetracycline antibiotics (Zhanel et al ., 2004 Agwuh and MacGowan, 2006 Hoffmann et al ., 2007 Barbour et al ., 2010).
We discussed above how metal ion binding adversely affects the absorption of tetracyclines from the GI tract, but it is an important and crucial part of the uptake of tetracyclines into bacterial cells. The chelation to a magnesium ion forms a tetracycline-magnesium cationic complex (with an overall charge of +1), which is transported through the outer membrane into the periplasm by porins, such as the well-characterised OmpF and OmpC examples from Gram negative bacteria (Nikaido, 1994, 2003). You will remember from Section 1.1.2 that porins are protein pores which transport a range of molecules through the outer membrane into the periplasm they are known to be responsible for the transport of other antibacterial agents, such as quinolones and β-lactams, besides tetracyclines (Jaffe et al ., 1982 Mortimer and Piddock, 1993). The mechanism for porin-mediated transport relies upon the Donnan potential 14 across the outer membrane and leads to accumulation of the tetracycline-magnesium cationic complex in the periplasm (Zhanel et al ., 2004). It is probable that the tetracycline-magnesium complex dissociates in the periplasm, perhaps due to the lower pH in this compartment, which is sufficiently acidic to drive the reprotonation of the enol oxygen at C12. The released zwitterionic tetracycline (overall neutral charge) is in equilibrium with a small proportion of the uncharged form, which is weakly lipophilic and able to diffuse through the cytoplasmic (inner) membrane in an energy-requiring process (Scheme 4.3.10) (Nikaido and Thanassi, 1993). Tetracyclines are presumed to adopt a similar uncharged tetracycline diffusion entry route through the simpler cell membrane of Gram positive bacteria.
Scheme 4.3.10 A tetracycline-magnesium complex dissociates to the zwitterion in the periplasm and equilibrates with the neutral form, which enters bacterial cells by passive diffusion (Nikaido and Thanassi, 1993)
You may be wondering why the diffusion of the neutral tetracycline across the bacterial cytoplasmic membrane is energy-requiring. Live bacteria have a difference in pH between the cytoplasm and periplasm of about 1.7 pH units (Nikaido and Thanassi, 1993), so that, after the neutral tetracycline passes through the cytoplasmic membrane, it becomes trapped at the higher pH of the cytoplasm, releasing a proton to form a greater proportion of tetracycline in a more hydrophilic form (overall molecular charge of −1) (Scheme 4.3.11). To maintain the pH of the cell, and the difference with the external pH, the bacteria must continue to transport protons out of the cytoplasm, which is an energy-dependent process (Nikaido and Thanassi, 1993), so it is not actually the diffusion of tetracycline into the cell that is energy-requiring, but the maintenance of pH that is required as a result.
Scheme 4.3.11 In the cytoplasm, the hydrophilic form of the tetracyclines predominates and reforms the tetracycline-magnesium complex (Nikaido and Thanassi, 1993)
The Effect of Kanamycin and Tetracycline on Growth and Photosynthetic Activity of Two Chlorophyte Algae
Antibiotics are routinely used in microalgae culture screening, stock culture maintenance, and genetic transformation. By studying the effect of antibiotics on microalgae growth, we can estimate the least value to inhibit growth of undesired pathogens in algal culture. We studied the effect of kanamycin and tetracycline on the growth and photosynthetic activity of two chlorophyte microalgae, Dictyosphaerium pulchellum and Micractinium pusillum. We measured CFU mL −1 on agar plates, optical density, fluorescence yields, and photosynthetic inhibition. Our results showed a significant effect of kan and tet on the tested microalgae species except tet, which showed a minor effect on M. pusillum. Both antibiotics are believed to interact with the protein synthesis machinery hence, the inhibitory effect of the tested antibiotics was further confirmed by isolation and quantification of the whole cell protein. A significant reduction in protein quantity was observed at concentrations more than 5 mg L −1 , except M. pusillum, which showed only a slight reduction in protein quantity even at the maximum tested concentration of tet (30 mg L −1 ). This study can further aid in aquaculture industry, for the maintenance of the microalgae stock cultures and it can also help the microalgae genetic engineers in the construction of molecular markers.
Microalgae are gaining importance in medical, pharmaceutical, and food industry. With the increasing applications of microalgae, it is mandatory to investigate growth conditions and potential growth inhibitors. Herbicides, antibiotics, and heavy metals are toxic to microalgae even at low concentrations [1–6]. Studying the survival and adoption of microalgae in the contaminated environment is not an insignificant question and to a certain extent, the microalgae could survive in contaminated environments [7–10].
In the past decade antibiotics use and resistance have been the focus of the world leading organizations, including the Center of Disease Control (CDC) and the World Health Organization (WHO). Alexander Fleming and Howard Walter Florey warned the world first time about the antibiotic resistance while receiving 1945 Nobel Prize for the discovery of penicillin . Antibiotic resistance has been a productive research topic for scientists in the medical field . Anthropogenic activities including use of antibiotics in agriculture, aquaculture, and waste disposal have been linked with the antibiotic resistance [13–15].
Aminoglycosides are the commonly used broad-spectrum antibiotics, that is, streptomycin, kanamycin, and amikacin. Aminoglycosides are characterized as multifunctional hydrophilic carbohydrates with several amino and hydroxyl activities having higher affinities to the prokaryotic rRNA [16, 17]. Suzuki et al. studied the effect of kanamycin on bacterial protein inhibition . Kestell et al. reported the effect of kanamycin and streptomycin on the macromolecular composition of Escherichia coli strains . The inhibitory effect of streptomycin had been reported to microalgae species at a concentration of 0.5 to 150 mg L −1 [20–22]. Galloway reported a halotolerant algae Amphora coffeaeformis resistance to streptomycin . Kvíderová and Henley reported the effect of ampicillin and streptomycin on the growth and photosynthetic activity of halotolerant chlorophyte algae species . However, a limited or no literature is available on the structural studies of aminoglycosides interaction with RNA sequences.
Kanamycin is a broad-spectrum aminoglycoside antibiotic, isolated from bacterium Streptomyces kanamyceticus . It is considered an important medication needed in a basic health system and it has been listed in the WHO’s list of Essential Medicines . Kanamycin interacts with the 30S ribosomal subunit resulting in a significant amount of mistranslation and prevents translocation during protein synthesis [27, 28], whereas tetracyclines bind to the 16S part of the 30S ribosomal subunit and prevent amino-acyl tRNA to attach at A-site of mRNA-ribosome complex, ultimately inhibiting protein synthesis as well as cell growth [29–31].
Kanamycin resistance (Kan R ) is mainly due to the cytoplasmic aminoglycoside phosphotransferase that inactivates kanamycin by covalent phosphorylation. On the other hand, tetracyclines are a group of broad-spectrum antibiotics, but their general application has been shortened because of the inception of antibiotic resistance [32–34]. Cells can become resistant to tetracyclines by one of the three mechanisms: enzymatic inactivation of tetracycline, efflux, and ribosomal protection .
Antibiotics tolerance of prokaryotic microorganisms has been described by leading scientists, but there are just a few reports available on the antibiotic tolerance study of eukaryotic microalgae [20, 22, 23, 36]. No doubt, antibiotics are normally considered effective against prokaryotic microorganisms, but they are extensively used in microalgae culture screening [37, 38], in aquaculture, and for screening of genetic transformants  hence, there is a need to check the effects of the antibiotics against eukaryotic microalgae.
This work was planned to determine the activity of two important antibiotics, kanamycin sulfate and tetracycline hydrochloride, against the freshwater eukaryotic microalgae species, Dictyosphaerium pulchellum and Micractinium pusillum. Colony forming units, optical density, fluorescence yields, and photosynthetic inhibitions were measured. The antibiotics used in this study are believed to interact with the protein synthesis machinery hence, the whole cell protein was also extracted and quantified.
2. Material and Methods
2.1. Microalgae Cultivation and Treatment
The eukaryotic freshwater microalgae species, Dictyosphaerium pulchellum and Micractinium pusillum, used in this study were obtained from the Korea Marine Microalgae Culture Center (KMMCC), Busan, South Korea. Stock cultures were stored on the modified AF6 agar slants . The cultures were streak plated and purified by subculturing by at least 5-6 times before use. Both microalgae species were cultivated in 250 mL flasks with 150 mL, modified AF6 medium while incubating at
μmol photons m −2 s −1 and 50% humidity. Antibiotics, kanamycin sulfate (Amresco), and tetracycline hydrochloride (Bio101) with different concentrations ranging from 0 to 30 mg L −1 were used. Growth rates were calculated by measuring the absorbance at 750 nm (OD750) on every alternating day . Additionally, all the experiments were repeated three times.
2.2. Screening Tests
The spread plate method according to Markham and Hagmeier , with slight modifications, was used to obtain colonies of the tested microalgae on agar plates. 200 μL of the cultured microalgae with approximately adjusted initial cell density (1 × 10 4 cells mL −1 ) was spread plated on AF6-agar plates supplemented with different concentrations of kan and tet ranging from 0 to 30 mg L −1 . Plates were incubated under constant light intensities and the growth was observed for three weeks.
2.3. Modulated Fluorescence and Photosynthetic Inhibition Measurement
Fluorescence yields of algae samples treated with different concentrations of kan and tet were measured by toxy-PAM dual channel yield analyzer (Heinz Walz GmbH, Effeltrich, Germany). The toxicity test is based on extremely sensitive measurement of the effective quantum yield (Y), of photosystem II (PSII), via assessment of chlorophyll fluorescence yield by following the saturation pulse method [43, 44]. Fluorescence of the dark adopted algal samples (
) is measured by using modulated light of low intensity to avoid the reduction of the PSII primary electron acceptor (
) . In order to induce an equilibrium state for the photosynthetic electron transport, prior to measurement of fluorescence, algal cells were adapted to darkness for 20 min.
In the toxy-PAM blue light is used for excitation and fluorescence is assessed at a wavelength above 650 nm. The ( ) fluorescence level corresponds to the fluorescence measured shortly before the application of a saturation pulse. Maximum fluorescence level (
) corresponds to the maximal fluorescence measured during a saturation pulse. The effective PSII overall quantum yield of the photochemical energy conversion was calculated by the formula given by Genty et al. .