Tetracyclines — extending the atypical spectrum

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International Journal of Antimicrobial Agents 3 (1993) S31-S46 © 1993 Elsevier Science Publishers BV 0924-8579/93/$24.00

ANTAGE 00094

Tetracyclines - extending the atypical spectrum G. Gialdroni Grassi Department of Chemotherapy, University olPavia, Pavia, Italy

(Accepted 25 August 1993)

The main feature and the present position of tetracyclines are revie~ved. The mechanism of their action, bacterialre istance alld the most recentfindings are reported. Their decreased use i due to the availability ofnew. active, better-tolerated antibiotics. However, tetracyclines still have a place il1 the treatment ofchlamydial and rickettsial infection, brucello is and Lyme disease. In respiratory infections, they can be employed ''v'hen necessar.v in infections caused b.v Chlamydia psittaci, C. pneumoniae, Mycopla rna pneumoniae, and also by Streptococcu pneurnoniae and Haemophilus influenzae, whose rates of resistance now seem lower than in the past when tetracyclines were more largely pre cribed.

Key words: Tetracyclines; Tetracycline analogue ; Antibiotic resistance; Spectrum of activity; lnfectiou diseases

Introduction Tetracyclines constitute a family of antibiotics, developed between 1948 and 1972, having in common a four-ring carboxylic structure. Differences between them are substitutions at position 5, 6 and 7 (Fig. 1). Naturally-occurring derivatives, such as chlortetracycline and oxytetracycline produced by Streptomyces aureofaciens and Streptomyces ri-

Correspondence to: Professor G. Gialdroni Grassi, Cattedra di Chemioterapia, Padiglione Forlanini, Via Taramelli 5, 27100 Pavia, Italy. Tel: +39382422233; Fax: +39382422267.

mosus, respectively, were followed by other derivatives, such as tetracycline, demethylchlortetracycline and subsequently by semisynthetic products, such as rnethacycline, doxycycline, rninocycline (also known as 'second-generation' tetracyclines) [1-3]. Tetracyclines were the first 'broad-spectrum antibiotics' and are active against a large number ofbacterial species including Gram-positive and Gramnegative organisms, some obligate anaerobes, Treponema pallidum, Coxiella burnetii, Chlamydia spp., Mycoplasma pneumoniae, Ureaplasma urealyticum and certain protozoa (Table 1) [4,5]. The enormous development of ~-lactam antibiotics (both penicillins and cephalosporins), with favourable characteristics of safety and which are en-

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Fig. 1. Fundamental structure of tetracycline derivatives showing the most important positions (5,6,7) for substitutions.

dowed with a broad antibacterial spectrum, permitted their use in the therapy ofa large numberofinfections that were previously treated with tetracyclines, On the other hand, erythromycin which was introduced into therapy in 1953 is active against pathogens such as Chlamydia, Mycoplasma and Legionella, against which p-Iactams are inactive, The availability of these and other antibiotics led to a gradual decline in the use of tetracyclines, to which, due to their previous widespread use, a large number of bacterial strains had become resistant. Some fastidious side-effects, such as brown discoloration of teeth, deposition in growing bone and hepatotoxic-

ity, further prevented the continued use of these widely applied drugs, The purpose of this paper is not to review all aspects of the activity and characteristics of tetracyclines that have been known for a long time, but to try to assess the present position and role of this class of antibiotics, Several changes in the aetiology of infections have occurred in the last few decades: new pathogens have been discovered and organisms previously considered as parasites are known to be causes of severe infections, It may be worthwhile, therefore, to reconsider the possible applications of tetracyclines in this context. Comprehensive reviews

S33 TABLE I Microorganisms sensitive to the tetracyclines Gram-positive organisms

Gram-negative organisms

Other organisms

Anaerobes

Staphylococcus aureus Streptococcus spp. Bacillus anthracis Corynebacterium diphtheriae Listeria monocytogenes Actinomyces israelii

Neisseria gonorrhoeae Neisseria meningitidis Enterobacteriaceae Haemophilus injiuenzae Flavohacterium tularensis Pasteurella multocida Vibrio cho/erae Bordetella pertussis Pseudomonas mallei Pseudomonas pseudomallei

Treponema pallidum Coxiella burnetii Rickettsia spp. Chlamydia spp. Mycoplasma hominis Mycoplasma pneumoniae Ureap/asma urea/yticum

Peptococcus spp. Peptostreptoccus spp. Propionibacterium spp. Clostridium spp.

on old and new acquisitions on tetracyclines have been recently published [6-9].

New tetracyclines Since the development of minocycline in 1972, no other tetracycline derivative has been introduced into

therapy. However, some new tetracycline analogues, e.g. chelocardin, 6-thiatetracycline and anhydrotetracyclines, have been developed with the aim of overcoming bacterial resistance to the earlier forms (Fig. 2) [9]. Even if due to toxicity [l 0, 11] none is clinically useful, the peculiar properties they show offer an interesting comparison with the older derivatives.

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Mechanism of action The classic mode of action of the early. tetracyclines is the inhibition of protein synthesis at the level of the ribosome. They reversibly bind to a single site of the 30S subunit, which explains their bacteriostatic activity [12,13]. Disruption of codon-anticodon interaction between transfer RNA and messenger RNA follows, so that binding to the ribosomal acceptor site is prevented. Using recently developed techniques, the region of tetracycline binding on the 30S ribosomal subunit has been determined; four proteins are present (S3, S7, S14 and S19) and direct binding of tetracycline to S7 protein has been demonstrated [14]. Besides the interactions with ribosomal proteins, tetracyclines can also indirectly interact with l6S ribosomal RNA through primary distortion of the ribosome [14-16]. The selectivity of action oftetracyclines for bacterial cells in comparison to eucaryotic cells is not solely due to a different affinity of the ribosome for these drugs. In fact, although the 80S ribosomes show a lower susceptibility to them [17], the mitochondrial 70S ribosomes behave in a similar manner to the bacterial ribosomes [18]. The main difference probably resides in the fact that the bacterial cell (but not the mammalian cell) actively concentrates tetracyclines [19]. The most prominent difference in the mode of action of early and new tetracyclines is that the latter are bactericidal, whereas the former are bacteriostatic [20,21]. Until recently the mechanism of action of 6-thiatetracyclines and analogues was attributed to ribosomal binding that probably differed from that of older tetracyclines [11,13]. Recent research, however, has demonstrated that the affinity of new derivatives for ribosomes is not sufficient to inhibit protein synthesis [16]. Their antibacterial activity has been attributed to their capacity to produce bacterial membrane damage; in fact, a profound inhibition of incorporation of precursors into macromolecules in the bacterial cell is observed [9,22,23]. The different mode of action is probably related to molecular conformation. While at physiological pH the early tetracyclines exist in an equilibrium mixture of two free-base forms: a low-energy lipophilic non-ionized form (for uptake across the cytoplasmic membrane) and a high-energy hydro-

philic zwitterionic structure (for binding to the ribosome) [11 ,24], the new derivatives, existing principally in the low-energy lipophilic form, are for the most part retained in the cytoplasmic membrane, where they cause irreversible damage [16,24].

Resistance to tetracyclines Some advances in the knowledge of the mechanisms of tetracycline resistance have been made in recent years [8]. The genes determining resistance to tetracyclines have been found in plasmids and transposons, and their spread to bacteria of different species and genera is the main factor responsible for the diffusion of high-level resistance. Resistance can be expressed through different biochemical mechanisms. Classically, it is assumed that while in susceptible bacterial cells there is an accumulation of tetracyclines, in resistant ones an energy-dependent efflux prevents accumulation of the drug in the cell. This mechanism of pumping out the drug is mediated by a resistance protein inserted into the bacterial cytoplasmic membrane. So far, this is the main mechanism of tetracycline resistance in Enterobacteriaceae, Vibrio, Aeromonas, Haemophilus, Pasteurella and Pseudomonas spp. Two new mechanisms of resistance have recently been discovered in other groups of bacteria (Fig. 3): namely, ribosomal protection whereby a cell component, presumably a protein, binds to the ribosome rendering it less susceptible to tetracyclines; and chemical modification of the tetracycline molecule determined by bacteria through an oxygen-requiring reaction, and rendering the drug inactive. The exact mechanisms of these two types of resistance have not yet been fully elucidated. In the case of ribosomal protection, two hypotheses have been put forward on the basis of some experimental findings. Resistance could be due to reduced binding of tetracyclines to the ribosome, or to weak binding to ribosomal sites other than the primary ones [25], probably because the distortion exerted by the resistant protein on the ribosome makes the secondary site more accessible. However, it has not been assessed whether the protein encoded by the resistance gene acts directly on ribosomes or modifies a host protein, to which the protective activity is devolved. There is

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Fig. 3. Schematic representation of the mode of action of tetracyclines in sensitive cells and the possible mechanisms of resistance [8,89]. (a) Tetracycline penetrates into sensitive bacterial cell, reaching much higher concentrations than the extracellular environment; it binds to the 30S ribosomal subunit inhibiting protein synthesis. (b) The classical mechanism of resistance; tetracyclines cannot reach a sufficient intracellular concentration due to an increased efflux. (c) Ribosomal protection mechanism of resistance; tetracyclines concentrate intracellularly, but ribosome is modified in such a way that the drug cannot bind effectively to it. (d) Tetracycline modification type of resistance; tetracyclines concentrate intracellularly but are rendered inactive by an oxygen-requiring chemical reaction. (Reproduced with permission from Salyers et al. [8]).

evidence of this type of resistance in Streptococcus, Campylobacter, Eikenella, Veillonella, Haemophilus, Gardnerella, Fusobacterium, Clostridium and Mycoplasma spp. [8]. The other type of resistance expressed as a modification to the tetracycline molecule is encoded by a gene present in two Bacteroides transposons that are also found in E. coli [26]. The gene product, a 44 kD cytoplasmic protein, detoxifies tetracycline by altering the antibiotic under aerobic conditions. The structure of the oxidized tetracycline has not been determined nor has it been established whether or not the tetracycline-modifying enzyme may cooperate in the naturally-occurring process of tetracycline auto-oxidation. This type of resistance is probably not a common natural occurrence, since the amount

of oxygen required for the reaction is higher than that usually encountered in body tissues [10,11].

Tetracycline pharmacokinetics Quite often data regarding the pharmacokinetic parameters of tetracycline derivatives are incomplete, probably because when they were developed less focus was placed on pharmacokinetic studies. The main pharmacokinetic features of these derivatives include adequate, but incomplete, absorption from the gastrointestinal tract, that can, however, reach 95% and 100% for doxycycline and minocycline, respectively [3,27-29]. The volume of distribution is larger than that of body water. These deriva-

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tives are concentrated in the liver and excreted through the bile, where they reach concentrations 5-20-fold higher than in plasma. Excretion takes place mainly through the kidney by glomerular filtration so that it may be greatly affected when renal function is impaired. Exceptions are represented by minocycline, which is considerably metabolized and excreted in lower amounts in urine and faeces, and by doxycycline that is scarcely eliminated by the renal route and can be administered in patients with renal failure. The serum protein binding ranges from about 30% for oxytetracycline to about 90% for doxycycline. The half-lives of tetracycline derivatives are shown in Table 2. The so-called 'secondgeneration' tetracyclines have longer half-lives than the earliest analogues (5-10 h) and it is questionable whether or not the dosing intervals prescribed (6~8 h) were adequate with the early analogues. The relatively low blood concentrations of tetracyclines may reflect a high degree of penetration into tissues. Accumulation in bone, chronically-inflamed tissues and tumoural cells has been demonstrated through the characteristic ultraviolet fluorescence of tetracyclines, and seems to support this hypothesis. Penetration of tetracyclines in cells has been studied in granulocytes [30,31], macrophages [30,32-34], erythrocytes [35] and tissue cells [30] with differing results. Intracellular:extracellular ratios usually range between 0.5 and 0.8, but higher values of up to 4.4 have been found in alveolar macrophages. Tetracyclines, therefore, do not concentrate in cells to the same extent as macrolides and clindamycin (intrace1TABLE 2 Half-lives of tetracyclines Half-lives (h) Tetracyclines Chlortetracycline Oxytetracycline Tetracycline Rolitetracycline Second-generation tetracyclines Demethylchlortetracycline Methacycline Doxycycline Minocycline

5-6 8-9.5 8-10

7-8

10-13 14

16--22 12-18

lular:extracellular ratio can be 10-20), nor does the uptake in macrophages of smokers increase as is the case for the above-mentioned antibiotics [36-38]. However, the concentrations that tetracyclines reach intracellularly assures their activity against intracellular pathogens [34,39], particularly Chlamydia and Rickettsia spp. (they are not active against Legionella). A pharmacokinetic parameter of some interest in the evaluation of possible tetracycline activity in respiratory infections is their concentration not only in the lung, but also in bronchial secretions. Here the concentrations of the majority of derivatives represent 12-20% of serum levels [40,41], whereas those of doxycycline and minocycline are 40-60% [40,42-45].

Practical uses of tetracyclines At present the clinical use of tetracyclines is limited due to the availability of a number of antibiotics with the same antibacterial spectrum and which are devoid of some fastidious side-effects, such as the discoloration of growing teeth and temporary inhibition of bone growth, which limit the use oftetracyclines in children and pregnant women. Moreover, tetracycline resistance is quite diffuse among some bacterial species of clinical interest. In addition to the clinical use in human medicine, tetracyclines are widely employed in veterinary medicine.

Use in veterinary medicine One ofthe main indications oftetracyclines in veterinary medicine is the treatment of infections due to Chlamydia psittaci in birds, particularly in psittacine species. The antibiotic can be added to the feed or water, but some precautions have to be taken to avoid binding to bivalent cations such as calcium and ferrous ions that may be present in feed and water, and which make the drug less available [4650]. Administration four to six times daily ofaerosolized tetracyclines revealed them to be effective in treating respiratory diseases [51,52]. Tetracyclines are widely used in ruminants for the treatment of tick-borne rickettsial infections: namely, anaplasmosis, heartwater and tick-borne fever [9]. The effectiveness of tetracyclines in the

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treatment of infections due to Anaplasma, particularly A. marginale, has been demonstrated since the early 1950s [53,54]. Cure is achieved when the treatment is started before the onset of anaemia and neurological signs [55]. The use of tetracycline also seems to be effective in the eradication of A. marginale in carriers [56,57]. Heartwater, a disease caused by Cowdria ruminantium, is widespread in tropical Africa and the West Indies. Tetracyclines represent the treatment of choice [58]. The disease can also be prevented by weekly injections for 3-4 weeks of long-acting oxytetracycline, administered just after exposure to the tick [59,60). Tick-borne fever is a disease caused by Cytoecetes phagocytophilia that is diffuse in northern Europe and India. Tetracyclines are used in the treatment of cows [61], and they are used prophylactically in lambs. A single dose of long-acting oxytetracycline has a prophylactic effect for about 15 days. If a short-acting preparation is employed, a reduction of morbidity and mortality is obtained. Moreover, since the microorganisms are not completely eliminated, the development of immunity can take place [61]. Tetracyclines have long been exploited as growth promoters in animals [62). The mechanism by which this effect is produced has not been completely elucidated, but it is likely to be due to modifications in the composition of the intestinal flora favouring the growth of the animal. This practice has been banned in several countries since it is considered potentially harmful, favouring the spread of resistant bacterial strains capable of producing difficult-to-treat diseases in humans. Opinion on this subject is still controversial, because indisputable proof of this assumption is not available [63,64]. Nevertheless, restrictions in this area of use seem wise.

Use in human medicine Despite some drawbacks in their use, tetracyclines still have a place in the treatment of a number of human infectious diseases. Sexually transmitted diseases. ChlamydiaI diseases

In recent years, non-gonococcal urethritis has been rapidly increasing in frequency. At present,

Chlamydia trachomatis is responsible for 30-50% or more of non-gonococcal urethritis in men. Moreover, chlamydial urethritis quite often follows a gonococcal infection both in men and women; sometimes it is asymptomatic and slowly self-limiting. Ureaplasma urealyticum belongs to the family Mycoplasmataceae and differs from genus Mycoplasma because of its capacity to split urea and form tiny colonies. It is only occasionally the cause of urethritis that is epidemiologically and clinically indistinguishable from that due to C. trachoma tis [65-69]. Some unusual immunotypes of C. trachomatis, defined as L-l, L-2 and L-3, are the aetiological agents of lymphogranuloma venereum, which is particularly widespread in south-east Asia and Africa. Clinical manifestations start with a non-indurated vesicular or papulovesicular lesion in the external genitalia, with regional adenopathy and systemic symptoms (fever, malaise, skin rashes, colitis, etc.) following after 1-2 weeks [68]. Granuloma inguinale is a sexually transmitted disease commonly occurring in tropical countries that is caused by a poorly characterized non-fermenting Gram-negative coccobacillus, Calymmatobacterium granulomatis. The papular lesions, which usually develop at the site of infection 2-3 months after exposure, gradually increase in size, giving rise to large granulomatous lesions [68]. For all the above described sexually transmitted diseases, tetracyclines are the treatment of choice [3,69,70]. In the past, they have often been prescribed in the knowledge that they are also effective against Neisseria gonorrhoeae, thus providing a therapy that covers both gonococcal and chlamydial infections. However, the increasing numbers and spread of resistant gonococcal strains now make this practice inadequate. Current treatment recommendations are to combine a single-dose of penicillin procaine (4.8 MU i.m.) or ampicillin (3 g p.o.), or alternatively (according to patient requirements and the extent of penicillin resistance in the area) ceftriaxone (250 mg i.m.) or spectinomycin (2 g i.m.), if gonococcal infection is suspected, followed by tetracycline (500 mg q.i.d. for 7 days) in order to treat the possible coexisting chlamydial infection [3,69,70]. Macrolides are also effective and are the treatment of choice in neonates to prevent and cure ophthalmia neonatorum, and to treat pneumonia ac-

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quired during parturition through the infected genital tract of the mother. Erythromycin has been largely used, although novel, better tolerated derivatives will probably contribute to the reduction in tetracycline usage [71]. Chlamydia trachomatis is also responsible for other ocular syndromes - trachoma and adult inclusion conjunctivitis. Trachoma is a chronic follicular keratoconjunctivitis, particularly diffuse in underdeveloped countries, where it is the major cause of blindness. Adult inclusion conjunctivitis is an acute follicular conjunctivitis, with a similar clinical picture to trachoma, but with an acute, benign course; however, if untreated, it may tend to become chronic. Treatment oftrachoma requires the use of a tetracycline derivative or sulphonamides for 3-6 weeks, but macrolides and rifampicin are also active. The same therapy is indicated for adult inclusion conjunctivitis, although usually for a shorter period of time. Erythromycin and tetracycline can also be applied to the eye as an ointment with concomitant oral treatment [68,69]. Another area in which Chlamydia spp. exert some important pathological roles is the respiratory system: C. trachomatis can be the cause ofpneumonia in neonates, whereas C. psittaci, C. pneumoniae and C. burnetii are responsible for pneumonia in adults (see Respiratory tract infections, page S40).

form which mimics sepsis or typhoid fever. Another form that is difficult to diagnose is the chronic form when it is not preceded by an acute phase. Due to the characteristics of the disease, therapy must be prolonged and aggressive in order to prevent relapses and complications. Tetracyclines are the drugs of first choice, with treatment lasting at least 3 weeks. Streptomycin has always been considered an effective companion drug for tetracycline, particularly in severe cases, in order to achieve eradication and avoid relapse [69,72]. A multicentre trial has shown that more prolonged treatment (45 days) with 200 mg/day doxycycline together with a single dose of 900 mg rifampicin, or with doxycycline plus streptomycin (l g i.m.) for the first 21 days produced cure rates of95% and 96%, respectively. These results are clearly better than those obtained with the currently proposed World Health Organization regimen of 2 g/day tetracycline for 21 days plus streptomycin (1 g/day i.m.) for 14 days, which gave a cure rate of59% [73]. So far, emergence of tetracycline-resistant Brucella spp. has not constituted a problem. In cases of intolerance to tetracyclines, alternative drugs are co-trimoxazole, in association with rifampicin or streptomycin, chloramphenicol and ciprofloxacin administered for at least 3 weeks. The macrolides, clarithromycin and azithromycin are more active than erythromycin against Brucella spp., whereas rifapentin has similar activity to rifampin [74].

Brucellosis Lyme disease and other borrelioses

Brucellosis is a serious illness with a widespread incidence, particularly in rural areas. Transmission takes place through contact with infected animals. No matter which species is the cause of the disease -Brucella melitensis from goats, B. abortus from cattle, B. suis from pigs - the clinical picture is the same but assumes many forms, so that it can mimic tuberculosis, malaria, typhoid fever, sepsis, etc. The most important feature of infections due to Brucella spp. is the invasion ofthe reticuloendothelial system. The intracellular localization of the microorganism and its capacity to survive within phagocytes are the features that lead to the chronicity of the disease [69,72]. Diagnosis of brucellosis is often difficult because of highly variable clinical manifestations and the delay in treatment can have serious consequences, even resulting in death in the hypertoxic

Lyme disease represents a new nosological entity.

It was recognized in the United States in 1977 after an outbreak of rashes and recurrent attacks of arthritis among children in Lyme, Connecticut [69,75,76]. The disease usually begins with erythema chronicum migrans (a syndrome that was described as long ago as 1910 in Europe) accompanied by headache, stiff neck, malaise, myalgias, arthralgias and swelling of the lymph nodes. After several weeks or months, symptoms involving the nervous system or heart can develop; meningoencephalitis or peripheral polyneuritis, myocarditis and migrating musculoskeletal pain are the most freq uent features of this state. These symptoms usually regress spontaneously in a few weeks.

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In about 60% of untreated patients, intermittent attacks of monoarticular arthritis of the large joints, particularly the knee, develop weeks or several months later. In about 10% of cases, arthritis can become chronic and refractory to therapy. The aetiological agent of the disease was discovered in 1981 - Borrelia burgdorferi, which belongs to the family Spirochetaceae and the order Spirochetales, and is transmitted by ticks of the genus Ixodes. The most frequently encountered species are I. dammini in central and eastern United States, whereas I. pacificus occurs in western United States and I. ricinus in Europe. Reservoirs of B. burgdorferi that infect ticks in nature are white-tailed deer, deer mice and the racoon. Other species of Borrelia infect poultry, other birds, horses and sheep. The progression of the human disease is not only related to the infectious agent, but also to a genetically determined host response, linked to class II major histocompatibility genes, in a position that evokes an autoimmune response after B. burgdorferi infection [69,76,77]. Borrelia burgdorferi is susceptible to many antibiotics, but tetracyclines seem to be the most clinically effective, particularly in the first stage of the disease [78,79]. Tetracycline at a dose of 500 mg q.i.d., or doxycycline (100 mg b.i.d.) when administered for 15-30 days can give excellent results. Some clinicians suggest that treatment should be extended for several months, but such practice, even if fairly widespread, is not supported by adequate controlled studies. Alternatives to tetracyclines are penicillin procaine, amoxycillin and erythromycin. Azithromycin, a novel azalide antibiotic, appears to be an effective alternative to erythromycin. In the second stage of the disease, penicillin G at a high dose of 14 g (given in divided doses), cefotaxime (3 g b.i.d.) and ceftriaxone (2 g/day) are the drugs most commonly employed; cephalosporins have been reported to produce better results than penicillin. The duration of treatment is usually 14-21 days. During the third stage, the choice is between ceftriaxone (or cefotaxime) and doxycycline. Optimal duration ofantibiotic therapy, however, has not yet been clearly assessed. Other species (Borrelia recurrentis, B. duttonii, B. persica, B. hispanica, B. turicatae, etc.) are responsible for relapsing fevers. B. recurrentis, which is transmitted by head and body lice, is widespread in Africa, and the other species are transmitted by ticks

found in the Americas, Africa, Asia and Europe [69]. The disease is characterized by high fever, chills, tachycardia, severe headache, vomiting, joint pains and, sometimes, an erythematous macular or purpuric rash, occasionally followed later by jaundice, hepatomegaly, splenomegaly and myocarditis. After 3-5 days, the fever declines after crisis, but it reappears a week or more later depending on the cyclic development ofthe parasites. Relapsing fever caused by any species of Borrelia can be treated with tetracyclines or, especially in children, with erythromycin or the newer macrolides [69,80]. In adults, a single 0.5 g oral dose oftetracycline or erythromycin cures louse-borne fever, whereas for tick-borne fever the full daily dosage of tetracycline or erythromycin (0.5 g q.i.d.) or doxycycline (l00 mg/day) must be administered for 5-10 days. Great care must be taken to prevent JarishHerxheimer reaction, which can easily occur due to rapid and massive destruction of microorganisms brought about by the antibiotics. Therapy should be started early in the febrile paroxysm or during the afebrile stage, with adequate measures being taken to avoid or reduce the severity of this side-effect (e.g. administration of an antipyretic, etc.). Melioidosis

Melioidosis is a disease caused by Pseudomonas pseudomallei, a Gram-negative aerobic bacillus that is a saprophyte of soil and water causing epizootic diseases in sheep, goats, pigs and horses. Humans contract melioidosis by contamination of skin abrasions. The disease is endemic in south-east Asia, although sporadic outbreaks have been reported in Africa, Turkey and Latin America. The most common form is an acute pulmonary infection of sudden onset, with consolidation of the superior lobes and often leading to cavitation. An acute septicaemic form can follow the pneumonia or can develop initially with severe general symptoms and often fatal outcome. The chronic suppurative form represents a chronic progression of the disease characterized by abscesses in the skin, lungs, brain, liver, spleen, bone,joints and lymph nodes. Exacerbations ofnonapparent or quiescent infection can take place several years after infection [68,69,81]. In pulmonary forms of the disease, drugs of first

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choice are tetracyclines (500 mg q.i.d. tetracycline or 100 mg b.i.d. doxycycline), followed by chloramphenicol, co-trimoxazole and ceftazidime [82] for a minimum of 1-5 months. For the treatment of more severe forms, a combination of tetracycline with cotrimoxazole or kanamycin for 30 days, followed by either tetracycline or co-trimoxazole for 1-3 months is suggested. For sepsis and the most severe forms, combined use of three antibiotics at high doses is recommended: tetracycline (80 mg/kg/day i.v. or p.o.), chloramphenicol (80 mg/kg/day i.v. or p.o.) plus co-trimoxazole (trimethoprim 9 mg/kg/day and sulphamethoxazole 45 mg/kg/day), or kanamycin (30 mg/kg/day), or novobiocin (60 mg/kg/day). This regimen should be continued until the patient is afebrile (but not for more than 1 week) and, thereafter, the dose should be suitably reduced. The reported efficacy of third-generation cephalosporins needs to be established more clearly using large-scale trials [68,69,81].

Respiratory tract infections Use of tetracyclines for the treatment of respiratory tract infections has declined, particularly in developed countries. The main reasons for this are the availability of new antimicrobial agents with excellent activity against respiratory pathogens together with improved tolerability and an increase in the incidence of resistance to tetracyclines, particularly in the case of Streptococcus pneumoniae and Haemophilus injluenzae, the two most important pathogens. Due to the decreasing use of tetracyclines in Western countries, it is probable that the incidence of resistant strains is now low [83]. Data from the UK indicate that the resistance rate of H. injluenzae was about 3% in 1977 and has not changed since then, although in some areas of the country figures of around 6-7% have been found [84,85]. In some respiratory infections, however, tetracyclines still have a role to play. In particular cases, therefore, tetracyclines can be employed with a chance ofgood clinical results and, in pneumonia due to P. pseudomallei, they are the drug of choice. The main indications of tetracyclines in lower respiratory tract infections are pneumonias due to Chlamydia or Rickettsia spp. Chlamydia psittaci is a parasite of birds and mammals, in which it can also pro-

voke a more or less severe disease, with humans acquiring C. psittaci usually as a result of inhalation of infected birds' excreta. The onset of pneumonia is abrupt, with fever, myalgias and headache, occasionally accompanied by hepatomegaly and splenomegaly, and followed by endocarditis [69,86]. Chlamydia pneumoniae is a recently discovered species (previously defined as TWAR) that causes a relatively mild respiratory disease usually with a long period of recovery [87]. Some syndromes, such as asthma, coronary artery disease, atherosclerosis, endocarditis, myocarditis, erythema nodosum and sarcoidosis have been associated with this infection, but the real value of these observations has to be confirmed. Chlamydia pneumoniae infections are both endemic and epidemic, and more than half of the adult population in the United States and in many other countries has antibodies to C. pneumoniae, an indication of past infection. Transmission seems to take place from human to human by respiratory tract secretions. Successful therapeutic results are obtained in pneumonia due to both Chlamydia spp. by giving tetracycline or doxycycline for 10-14 days. Erythromycin is also active, and clarithromycin and azithromycin are highly active in vitro. Due to their improved pharmacokinetic properties, the latter agents will probably replace the older macrolides. The efficacy ofthe new fluoroquinolones requires further studies. Coxiella hurnerii, a rickettsial species responsible for Q fever, has reservoirs in cattle, goats and ticks; thus, workers involved in slaughtering and the processing of infected animals are at high risk. Infection can be mild or non-apparent, but when it develops the onset is sudden with high fever and a series of general, non-specific clinical manifestations. The frequency of pneumonia in infected patients varies from region to region and may be as high as 90% of cases in some settings [69,86,88]. Tetracycline (500 mg q.i.d.) and doxycycline (100 mg b.i.d.) are the drugs of choice. Chloramphenicol and macrolides can also be effective, but the role of the new fluoroquinolones has yet to be assessed. Mycoplasma pneumoniae is the aetiological agent of pneumonia often known as primary atypical pneumonia that predominantly occurs in people aged between 5 and 30 years, although there appears to be an increase in the frequency of the disease in

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older subjects. The onset and the course of the disease are quite insidious and resolution is slow [69,86]. Tetracyclines and macrolides are equally effective against mycoplasmal pneumonia; however, macrolides are preferable for the treatment of children, because ofthe well-known tendency oftetracycline to be incorporated into growing bone and teeth causing a brown discoloration of the latter. New macrolide derivatives and fluoroquinolones are also of value in the treatment of mycoplasmal pneumonia. An additional area of respiratory infections in which tetracyclines can be employed is that related to anaerobic infection (necrotizing pneumonia, lung abscesses, empyema). Among the tetracyclines, doxycycline and minocycline are the most active; however, many strains, particularly those belonging to Bacteroides spp., are resistant, so that metronidazole, clindamycin, cefoxitin and cefotetan are preferred. Chloramphenicol has good activity against all anaerobes. In severe cases, because most infections are due to mixed flora, broad-spectrum antibiotics such as intravenous imipenem, piperacillin, ticarcillin plus clavulanic acid, chloramphenicol and penicillin G at very high doses, are given in association with metronidazole [86]. Miscellaneous uses

Systemic therapy of acne and rosacea with tetracyclines has produced very favourable results, probably as a consequence of the activity of these antibiotics on Propionibacterium acnes, an anaerobic microorganism producing lipase at skin level. This feature leads to reduced free fatty acids, thus promoting inflammation. The suggested dosage for tetracycline is 0.25-0.5 mg b.i.d., to be reduced by 50% when improvement is achieved; however, minocycline (100 mg/day) seems to be more active but the duration of therapy has not been clearly established. It seems advisable to stop treatment periodically (perhaps at monthly intervals) to check if remission has been reached. At present, topical application of clindamycin is also widely used [3,89,90]. Use of tetracyclines in the treatment of gastrointestinal infections is declining, even though, in developing countries they are frequently used for economic reasons. Shigellosis and non-typhoidal salmonellosis may not respond to treatment with tetra-

cyclines, since most strains are resistant, but in some instances they can still be effective. Some diarrhoeal diseases caused by Escherichia coli, which above all should be treated by replacement of fluid and electrolyte losses, can be shortened by the administration of tetracycline or co-trimoxazole [68,69]. Cholera can also be cured by simple replacement oflosses ofliquid and electrolytes, but it is also susceptible to treatment with tetracycline (250-500 mg q.i.d. for 2 days). At present many strains of Vibrio cholerae in Bangladesh and Tanzania are tetracycline-resistant [69,91]. Fluoroquinolones, however, are efficacious in the treatment of gastrointestinal infections resistant to other antibiotics [69,92]. There is a group of rickettsial diseases known as typhus fevers. Rickettsia prowazekii, the cause of louse-borne and squirrel-borne typhus, and Rickettsia typhi, the cause of flea-borne or endemic typhus, are among the most common pathogens, but have different geographical distributions. The early symptoms of the diseases are similar: fever, headache and myalgias. Diagnosis can be difficult due to the many forms the disease can take and the course of the disease is sometimes severe. Tetracyclines and chloramphenicol are the drugs of choice, but fluoroquinolones are also effective. Treatment can be continued for 2-5 days after defervescence. Another group of rickettsial species (Rickettsia rickettsii, R. conorii, R. sibirica and R. australis) is the cause of so-called spotted fever, characterized by a black spot or eschar at the site of the tick bite. This is accompanied by fever, lymphadenopathy, headache, myalgias and a generalized macopapular rash but the course of the disease is seldom severe and it responds promptly to tetracycline, doxycycline and chloramphenicol, and also to fluoroquinolones (ciprofloxacin and ofloxacin). Therapy usually lasts a few days, although I-day treatment with doxycycline may be sufficient for the treatment of Mediterranean spotted fever [93]. Scrub typhus, due to Rickettsia tsutsugamushi, is transmitted to humans via the bite of some infected larval trombiculid mites found in south-east Asia, Japan and the western Pacific islands [69]. The illness has an insidious feverish onset and a distinctive feature is the presence of a black eschar, which forms at the site where the chigger was attached, but it is apparent in only about 50% of patients. Tetracycline (500 mg

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q.i.d. for 7 days) or doxycycline (200 mg as a single dose) or chloramphenicol (500 mg q.i.d. for 7 days) brings about a prompt resolution of symptoms. Rickettsial pox is due to Rickettsia akari, which is transmitted to humans by the bite of Lypnissoides sanguineus, a mite whose host is the house mouse. The initial manifestation of the disease is a papule at the site of the mite's bite, which is followed in about 10 days by enlargement of regional lymph nodes, malaise, fever and myalgias; and thereafter, more severe symptoms and involvement may evolve. Tetracycline (250-500 mg q.i.d.) or doxycycline (lOO mg b.i.d.), administered for 2-5 days, is efficacious. Chloramphenicol is the alternative drug [68,69]. Other uses of tetracyclines include the frequent treatment of dental infections, efficacy being probably attributable not only to their activity against oral bacteria, but also to their good distribution in oral tissues [94]. In leptospirosis, doxycycline (100 mg b.i.d. for I week) can shorten the course of early disease [95]. Furthermore, recent studies suggest the administration of tetracycline for the treatment ofbacilIary angiomatosis, a syndrome with cutaneous lesions indistinguishable from Kaposi's sarcoma, or pyogenic granuloma, which occurs most frequently in HIV-infected patients [96,97]. The microorganism responsible seems to be Rochalimaea quintana which, using hybridization techniques, appears to share some similarities with the agent responsible for cat-scratch disease (Ajipia felis). Like the latter, R. quintana is not susceptible to penicillin, nafcillin, dicloxacillin, or cephradine, but unlike A. felis, it responds to doxycycline, erythromycin and rifampin. In 3-4 days, clinical symptoms subside, while laboratory parameters are normalized in about 2 months. Possible new role of tetracyclines A new role for tetracyclines is found in human immunodeficiency virus (HIV) infection. According to certain studies [98-100], supported also by some of the findings of Montagnier et al. [l 0 1], certain Mycoplasma spp. (M. incognitum or fermentans, M. pirum, M. genitalium) act as cofactors in AIDS pathogenesis. They stimulate T-lymphocytes and penetrate HIV-infectedcells, favouring HIV replication and enhancing the HIV cytopathic effect. Proof

of the activity of Mycoplasma species resides in the demonstration that antibodies directed against a peptidic sequence common to M. genitalium and M. pirum inhibit HIV replication and pathogenicity [101]. Tetracycline derivatives show a protective role in vitro against the cytopathic effect of HIV in CEM cell cultures (a T-Iymphoblastoid tumour cell line), but not against virus growth. It is suggested that cytopathogenicity of HIV may be due to the presence of tetracycline-susceptible Mycoplasma in the cell [102]; therefore, tetracycline activity against Mycoplasma could indirectly contribute to the inhibition of certain steps in the pathogenic pathway of HIV infection. This hypothesis is still controversial and its real value has to be assessed with further more convincing demonstrations.

Conclusion The family of tetracycline antibiotics has not shown any impressive development in recent years, contrary to what has been observed in other groups of antibiotics, such as ~-lactams. A reduction in tetracycline use has been a common feature in developed countries, particularly due to the availability of a large number of antibiotics with excellent antibacterial activity and good tolerability. However, tetracyclines maintain their value in the treatment of many infections in veterinary medicine. In human medicine, they also remain drugs of first choice for many important infections: non-gonococcal urethritis; brucellosis; Lyme disease and other borrelioses; and rickettsial diseases, such as typhus fever and other spotted fevers. In addition, they can be usefully employed, if necessary, in gastrointestinal infections, dental infections, leptospirosis and acne. In respiratory infections they are the drug of choice in melioidosis and maintain an excellent activity against intracellular pathogens, particularly Chlamydia and Rickettsia species. The second-generation tetracyclines, whose development started in the early 1980s, are endowed with the very interesting characteristics of being bactericidal, having a peculiar activity on the bacterial cytoplasmic membrane. Owing to their toxicity, none of these analogues has so far found any clinical application; future developments in this area are possible.

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References

2

3

4

5

6

7 8 9

10 II

12

13

14

15

16

17

Valcavi U. Tetracyclines: chemical aspects and some structure-activity relationships. In: Grassi GG, Sabath LD, eds. New Trends in Antibiotics: Research and Therapy. Amsterdam: ElsevierlNorth-Holland Biomedical Press, 1981. Col NF, O'Connor RW. Estimating worldwide current antibiotic usage: report ofTask Force I. Rev Infect Dis 1987;9 (suppl 3):S232-S243. Sande MA, Mandell GL. Tetracyclines, chloramphenicol, erythromycin and miscellaneous antibacterial agents. In: Goodman Gilman A, Goodman LS, Rail TW, Murad F, eds. The Pharmacological Basis of Therapeutics, 7th edn. New York: Macmillan, 1985. Cunha BA. Clinical uses of the tetracyclines. In: Hlavka 11, Boothe lH, eds. The Tetracyclines. Berlin: Springer-Verlag, 1985. Edlin TO. Tetracyclines as antiparasitic agents: lipophilic derivatives are highly active against Giardia lamblia in vitro. Antimicrob Agents Chemother 1989;33:2144-2145. Chopra I, Howe TGB, Linton AH, Linton KB, Richmond MH, Speller DCE. The tetracyclines: prospects at the beginningofthe 1980s. 1 AntimicrobChemother 1981;8:5-21. Hlavka 11, Boothe lH, eds. The Tetracyclines. Berlin: Springer-Verlag, 1985. Salyers AA, Speer BS, Shoemaker NB. New perspectives in tetracycline resistance. Mol Microbiol 1990;4: 151-156. Chopra 1, Hawkey PM, Hinton M. Tetracyclines, molecular and clinical aspects. 1 Antimicrob Chemother 1992;29:245-277. Mitscher LA. The Chemistry of the Tetracycline Antibiotics. New York: Marcel Dekker, 1987. Rogalski W. Chemical modification of tetracyclines. In: Hlavka 11, Boothe lH, eds. The Tetracyclines. Berlin: Springer-Verlag, 1985. Gale EF, Cundliffe E, Reynolds PE, Richmond MH, Waring MJ. The Molecular Basis of AntibioticAction. London: lohn Wiley, 1981. Chopra I. Mode ofaction of the tetracyclines and the nature of bacterial resistance to them. In: Hlavka 11, Boothe lH, eds. The Tetracyclines. Berlin: Springer-Verlag, 1985. Buck MA. Cooperman BS. Single protein omission reconstitution studies of tetracycline binding to the 30S subunits of Escherichia coli ribosomes. Biochemistry 1990;29:53745379. Moazer 0, Noller HF. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature 1987;327:389394. Rasmussen B, Noller HF, Daubresee G et al. Molecular basis of tetracycline action: identification of analogs whose primary target is not the bacterial ribosome. Antimicrob Agents Chemother 1991 ;35:2306-2311. Van den Bogert C. Kroon AM. Tissue distribution and effects on mitochondrial protein synthesis of tetracyclines

18

19

20

21 22

23 24

25

26

27

28

29 30

31

32

33

34

after prolonged continuous intravenous administration to rats. Biochem Pharmacol 1981 ;30: 1706-1709. Riesbeck K, Bredberg A, Forsgren A. Ciprofloxacin does not inhibit mitochondrial functions but other antibiotics do. Antimicrob Agents Chernother 1990;34:167-169. Franklin TJ. Mode of action of the tetracyclines. In: Newton BA, Reynolds PE, eds. Biochemical Studies of Antimicrobial Drugs: Sixteenth Symposium ofSociety for General Microbiology. Cambridge: Cambridge University Press, 1966. Proctor R, Craig W, Kunin C. Cetocycline, tetracycline analog: in vitro studies of antimicrobial activity, serum binding, lipid solubility, and uptake by bacteria. Antimicrob Agents Chemother 1978; 13:598-604. Bakhtiar M, Selwin S. Antibacterial activity of a new tetracycline. 1 Antimicrob Chemother 1983;11:291. Sahl HG. Influence of the staphylococcin-like peptide Pep 5 on membrane potential of bacterial cells and cytoplasmic membrane vesicles. 1 Bacteriol 1985; 162:833-836. Russell AD, Chopra I. Understanding antibacterial action and resistance. London: Ellis Horwood, 1990. Hughes LJ, Stezowski 11, Hughes RE. Chemical-structural properties of tetracycline derivatives. 7. Evidence for the coexistence ofthezwitterionic and non-ionized forms of the free base in solution. 1 Am Chern Soc 1979; 101 :7655-7656. Goldman RA. Photoincorporation of tetracycline into E. coli ribosomes. Identification of the major proteins photolabeled by native tetracycline and tetracycline photo products and implications for the inhibitory action of tetracycline on protein synthesis. Biochemistry 1983;22:359-368. Speer BS, Salyers AA. Characterization of a novel tetracycline resistance that functions only in aerobially grown Escherichia coli. 1 BacterioI1988;170:1423-1429. Spitzy RH. Tetracyclines. In: Kuemmerle HP, ed. Clinical Chemotherapy, Vol II. Antimicrobial Chemotherapy. New York: Thieme Stratton, 1983. Saivin S, Houin G. Clinical pharmacokinetics of doxycycline and minocycline. Clin Pharmacokinet 1988;15:355366. Gialdroni-Grassi G. Chemioantibioticoterapia. Milan: Masson, 1989. Brown KN, Percival A. Penetration of antimicrobials into tissue cells and leukocytes. Scand 1 Infect Dis [Suppl] 1978; 14:251-260. Park YK, Dow RC. The uptake and localization oftetracycline in human blood cells. Br 1 Exp Pathol 1970;51: 179182. lohnson 10, Hand WL, Francis lB, King-Thompson N, Corwin RW. Antibiotic uptake by alveolar macrophages. 1 Lab Clin Med 1980;95:429--439. Hand Lee W, Corwin RW, Steinberg TN, Grossman GO. Uptake ofantibiotics by human alveolar macrophages. Am Rev Respir Dis 1984;129:933-937. Ekzemplyarov ON. Penetration oftetracycline and strepto-

S44

35

36

37

38 39

40

41 42

43 44

45

46

47

48

49

50

51 52

mycin into macrophages cultured in vitro. Antibiotiki 1965; 10:Tl32-T314. Kornguth ML, Kunin CM. Binding of antibiotics to the human intracellular erythrocyte proteins hemoglobin and carbonic anhydrase. J Infect Dis 1976;133:185-193. Hand WL, Boozer RM, King-Thompson WL. Antibiotic uptake by alveolar macrophages of smokers. Antimicrob Agents Chemother 1985;27:42-45. Carlier MB, Zenebergh A, Tulkens PM. Cellular uptake and subcellular distribution of roxithromycin and erythromycin in phagocytic cells. J Antimicrob Chemother 1987;20 (suppl B):47-56. Tulkens PM. Intracellular distribution and activity of antibiotics. Eur J Clin Microbiol Infect Dis 1991;10:100-106. Richardson M, Holt IN. Synergistic action of streptomycin with other antibiotics on intracellular Brucella abortus in vitro. J BacterioI1962;84:638-646. McArthur CGC, Johnson AJ, Chadwick MV, Wingfield HJ. The absorption and sputum penetration of doxycycline. J Antimicrob Chemother 1978;4:509-514. Campbell MJ. Tetracycline levels in bronchial secretions. J Clin PathoI1970;23:427-434. Marschang A, Diezel PB, Klein G. Die Konzentrationen von Doxycyclin in Blutserum und Bronchialsekretion. Prax Klin PneumoI1978;32:271-273. Hartnett BJS, Marlin GE. Doxycycline in serum and bronchial secretions. Thorax 1976;31:144-148. Bregan TD, Neale L, Ryley HC, Davies BH, Charles J. The secretion of minocycline ill sputum during therapy of bronchopulmonary infection in chronic chest diseases. J Antimicrob Chemother 1977;3:247-251. Bergogne-Berezin E, Lambert-Zechovsky N, Morel e. Pharmacocinetique des antibiotiques dans les secretions bronchiques. Nouv Presse Med 1977;6:35-48. Clary BD, Terry RJ, Creger CR. The potentiation effect of citric acid on aureomycin in turkeys. Poult Sci 1981 ;60: 1209-1212. Pollet RA, Glatz CE, Dyer DC, Barnes HJ. Pharmacokinetics of chlortetracycline potentiation with citric acid in the chicken. Am J Vet Res 1983;44:1718-1721. Pollet RA, Glatz CE, Dyer De. Oral absorption of chlortetracycline in turkeys; influence of citric acid. Poult Sci 1984;63:1110-1114. Schachter J, Bankowski RA, Sung ML, Miers L, Strassburger M. Measurement of tetracycline levels in parakeets. Avian Dis 1984;28:490-495. Flammer K, Cassidy DR, LandgrafWW, Ross PF. Blood concentrations of chlortetracycline in macaws fed medicated pelleted feed. Avian Dis 1989;33: 199-203. Ficken MD. Antibiotic aerosolization for treatment of Alcaligenes rhinotracheitis. Avian Dis 1983;27:545-548. Dyer DC, Van Alstine WG. Antibiotic aerosolization: tissue and plasma oxytetracycline concentration in parakeets. Avian Dis 1987;31:677-679.

53 Magonigle RA, Simpson JE, Frank FW. Efficacy of a new oxytetracycline formulation against clinical anaplasmosis. Am J Vet Res 1978;39:1407-1410. 54 Kuttler KL. Pharmacotherapeutics of drugs used in treatment of anaplasmosis and babesiosis. J Am Vet Med Assoc 1980;176:1103-1108. 55 Magonigle RA, Newby TJ. Elimination of naturally acquired chronic Anaplasma marginale infections with longacting oxytetracycline injectable. Am J Vet Res 1982;43:2170-2172. 56 Splitter EJ, Miller JG. The apparent eradication of the anaplasmosis carrier state with antibiotics. Vet Med 1953;48:486-488. 57 Rogers RJ, Dunster PJ. The elimination of Anaplasma marginale from carrier cattle by treatment with long-acting oxytetracycline. Aust Vet J 1984;61 :306. 58 Haig DA, Alexander RA, Weiss KE. Treatment ofheartwater with terramycin. J S Afr Vet Med Assoc 1954;24:45-48. 59 Purnell RE. Development of a prophylactic regime using Terramycin/LA to assist in the introduction of susceptible cattle into heartwater endemic areas of Africa. OnderstepoortJ Vet Res 1987;54:509-512. 60 Purnell RE, Gunter TD, Shroder J. Development of a prophylactic regime using long-acting tetracycline for the control of redwater and heartwater in susceptible cattle moved in to an endemic area. Trop Anim Health Prod 1989;21:11-19. 61 Brodie TA, Holmes PH, Urquhart GM. Prophylactic use of long-acting tetracycline against tick-borne fever (Cytoecetes phagocytophilia) in sheep. Vet Rec 1988;15:161202. 62 Visek WJ. The mode of growth promotion by antibiotics. J Anim Sci 1978;46:1447-1469. 63 Budiansky S. Jumping the smoking gun. Nature 1984;311 :407. 64 Holmberg SD, Osterholm MT, Senger KA, Cohen ML. Drug-resistant Salmonella from animals fed antimicrobials. N Engl J Med 1984;311 :617-622. 65 Volk J, Kraus SJ. Non-gonococcal urethritis: a venereal disease as prevalent as epidemic gonorrhoea. Arch Intern Med 1974;134:511. 66 Zimmerman HL, Potterat JJ, Dukes RL, Muth JB, Zimmerman HP, Pratts CI. Epidemiologic differences between chlamydia and gonorrhoea. Am J Public Health 1990;80: 1338-1342. 67 Schachter J. Chlamydial infections. N Engl J Med 1978;28:490-495. 68 Reese RE, Douglas RG Jr, eds. A Practical Approach to Infectious Diseases. Boston/Toronto: Little, Brown, 1986. 69 Hoeprich PD, Jordan Me. Infectious Diseases, 4th edn. Philadelphia: JB Lippincott, 1988. 70 Centers for Disease Control. 1989 sexually transmitted diseases treatment guidelines. MMWR Morb Mortal Wkly Rep 1989;38(8):21-27.

S45 71 Peters DH, Heather A, McTavish F, McTavish D. Azithromycin: a review of its antimicrobial activity, pharmacokinetic properties and clinical efficacy. Drugs 1992;44:750799. 72 Young EJ. Human brucellosis. Rev Infect Dis 1983;5:821842. 73 Acocella G, Berttrand A, Boytout J et al. Comparison of three different regimens in the treatment of acute brucellosis: a multicentre multinational study. J Antimicrob Chemother 1989;23:433--439. 74 Garcia Rodriguez JA, Munoz Bellido YL, Fresnadillo MJ, Trufyilland 1. In vitro activities of new macroIides and rifapentine against Brucella spp. Antimicrob Agents Chemother 1993;37:911--913. 75 Lennette EH, Balows A, Hausler WJ, Shadomy HJ. Manual of Clinical Microbiology, 4th edn. Washington DC: American Society for Microbiology, 1985. 76 Schoen RT. Lyme disease. Curr Opin Infect Dis 1991 ;4:609-614. 77 Steere AC, Dwyer E, Winchester R. Association ofchronic Lyme arthritis with HLA-DR4 and HLA-DR2 alleles. N Engl J Med 1990:323:219-223. 78 Luft HJ, Gorevic PD, Halperin JJ, Volkman DJ, Dattwyler RJ. A perspective on the treatment ofLyme borreliosis. Rev Infect Dis 1989;11 (suppI6):1518-1525. 79 Sigal LH. Current recommendation for the treatment of Lyme disease. Drugs 1992;43:683-699. 80 Galun E, Ben-Chetri E. Possible prevention of tick-borne relapsing fever in patients infected with Borrelia recurrentis. lInfectDis 1984;150:617. 81 Dance DAB. Melioidosis: the tip of the iceberg? Clin Microbioi Rev 1991;4:52-60. 82 White NJ, Chaowagul W, Wuthiekanun V, Dane DAB, Wattanagoon Y, Pitakwatchara N. Halving of mortality of severe melioidosis by ceftazidime. Lancet I989;i:697-700. 83 Maesen FPV, Davies BI, Van den Berg JJAM. Doxycycline and minocycline in the treatment of respiratory infections: a double-blind comparative clinical, microbiological and pharmacokinetic study. J Antimicrob Chemother 1989;32: 123-129. 84 Powell M, Koutsia-Carouzou C, Voulsinas D, Seymour A, Williams JD. Resistance of clinical isolates of Haemophilus injluenzae in United Kingdom 1986. Br Med J 1987;295: 176--179. 85 Research Committee of the British Thoracic Society and the Public Health Laboratory Service. Community-acquired pneumonia in adults in British hospitals in 19821983: a survey of aetiology, mortality, prognostic factors and outcome. Q J Med 1987;62: 195-220. 86 Pennington JE. Respiratory Infections: Diagnosis and Management, 2nd edn. New York: Raven Press, 1989. 87 Grayston JT. Infection caused by Chlamydia pneumoniae strains TWAR. Clin Infect Dis 1992;15:757-763. 88 Sawyer LT, Fishbein DB, McDadeJE. Qfever: current concepts. Rev Infect Dis 1987;9:935-946.

89 Cunliffe WJ. Treatment of acne. In: Marks R, Plewig G, eds. Acne and Related Disorders. London: Martin Dunitz, 1989. 90 Eady EA, Cove JH, Holland KT, Cunliffe WJ. Superior antibacterial action and reduced incidence of bacterial resistance in rninocycline compared to tetracycline-treated acne patients. Br J DermatoI1990;122:233-244. 91 Alam AN, Alam NH, Ahmed T, Sack DA. Randomized, double blind trial of single dose doxycycline for treating cholera in adults. Br Med J 1990;300: 1619-1621. 92 Wolfson JS, Hooper DC. Quinolone Antimicrobial Agents. Washington DC: American Society for Microbiology, 1989. 93 Bella-Cueto F, Font-Crues B, Segura-Porta F, Espejo-Arenas E, Lopez-Pers P, Munoz-Espin T. Comparative, randomized trial ofone-day doxycycline versus lO-day tetracycline therapy for Mediterranean spotted fever. J Infect Dis 1987;155: 1056--1058. 94 Wade WG. In-vitro activity of ciprofloxacin and other agents against oral bacteria. J Antimicrob Chemother 1989;24:683-687. 95 Watt G. Leptospirosis. Curr Opin Infect Dis 1992:5:659663. 96 ChanJK, Lewin KJ, LombardCM, TeitebaumS, Dorfman RF. Histopathology of bacillary angiomatosis of lymph node. Am J Surg PathoI1991;15:430--437. 97 Hadfield TL. Cat-scratch disease and bacillary angiomatosis. Curr Opin Infect Dis 1992;4:628-635. 98 Chaodury IH, Munakata T, Koyanagi Y, Kobayashi S, Arai S, Yamamoto N. Mycoplasma can enhance HIV replication in vitro: a possible co-factor responsible for the progression of AIDS. Biochem Biophys Res Commun 1990; 170: 1365-1370. 99 Lo S, Tsai S, Benisk JR, Shih JWK, Wear DJ, Wong DM. Enhancement of HIV-I cytocidal effects in CD4 lymphocytes by the AIDS-associated Mycoplasma. Science 1991 ;251: 1074-1076. 100 Baseman JB, Quackenbush RL. Preliminary assessment of AIDS associated Mycoplasma. ASM News 1990;56:319323. 101 Montagnier L, Barneman D, Guetard D et al. Inhibition de I'infectiosite de souches prototypes du HIV par des anticorps diriges contre une sequence peptidique de Mycoplasme. C R Acad Sci Paris 1990;311 :425--430. 102 Lemaitre M, Guetard D, Hemin Y, Montagnier L, Zrial A. Protective activity of tetracycline analogues against the cytopathic effect of the human immunodeficiency viruses in CEM cells. Res ViroI1990;141:5-16.

Discussion H Giamarellou (Greece): Do you suggest that up until now we have been incorrectly prescribing tetra-

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cyclines? You pointed out the prolonged half-life, yet most tetracyclines are prescribed every 6 hand doxycycline every 12 h. G Gialdroni Grassi (Italy): I meant that, considering the half-lives of tetracycline derivatives in the

light of present knowledge, the 6-h interval, which was chosen when tetracyclines were first introduced, seems to be short. I think that doxycycline is prescribed in the correct way now. The other compounds are probably given at too short intervals.