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Copyright ERS Journals Ltd 1994 European Respiratory Journal ISSN 0903 - 1936 Eur Respir J, 1994, 7, 247–250 DOI: 10.1183/09031936.94.07020247 Printed in UK - all rights reserved Mycobacterium avium complex develop resistance to synergistically active drug combinations during infection S.E. Hoffner*, N. Heurlin**, B. Petrini†, S.B. Svenson*, G. Källenius* Mycobacterium avium complex develop resistance to synergistically active drug combinations during infection. S.E. Hoffner, N. Heurlin, B. Petrini, S.B. Svenson, G. Källenius. ERS Journals Ltd 1994. ABSTRACT: Isolates of Mycobacterium avium complex from five patients on longterm (3–5 yrs) anti-mycobacterial drug treatment were collected during the early and late phase of disease, and studied in vitro for their susceptibility to anti-mycobacterial drugs and drug-combinations. All isolates were resistant or moderately resistant to ethambutol, rifampicin and streptomycin when given singly; however, all strains isolated early in the disease were susceptible to the combination of ethambutol with either rifampicin or streptomycin. All late isolates had developed resistance to one or both of these combinations. Three of the patients died within a year after the last isolation of M. avium complex, and the two remaining patients still have severe chronic disease. It is concluded that the susceptibility of M. avium strains to combinations of drugs should be monitored during the course of treatment, in order to guide the selection of effective drug-combinations throughout the infection. Eur Respir J., 1994, 7, 247–250. Infections caused by bacteria belonging to the Mycobacterium avium complex (MAC) are increasingly recognized as clinically important. MAC is, after M. tuberculosis, the most common cause of mycobacterial pulmonary disease. In addition, severe MAC infections are common in patients with acquired immune deficiency syndrome (AIDS) [1, 2]. Infections caused by MAC are generally regarded as difficult to treat. However, with the drug combinations in use at the present time, a considerable number of patients are successfully cured. This is true, in particular, for patients with pulmonary MAC infections and without serious predisposing factors. On the other hand, there are many patients who, although the bacterial load can often be significantly reduced, may never be cleared from the infecting mycobacteria. This is often seen in patients suffering from severe immunodeficiencies, and especially in human immunodeficiency virus (HIV)-infected patients. Most MAC strains are highly resistant to anti-mycobacterial drugs in vitro when given singly. Hence, much of the blame for treatment failures of MAC infections has been ascribed to the inherent drug resistance. However, most clinical isolates of MAC are susceptible to certain combinations of drugs [3–8]. These findings correspond to in vivo observations that the same drug combinations are, in many cases, quite effective against disease caused by MAC [9, 10]. However, the effect of such combined drug treatment is sometimes only temporary. One reason for this may be the development of resistance to initially effective combinations of drugs. We therefore studied the susceptibility to the two-drug combinations, *Dept of Bacteriology, National Bacteriological Laboratory, Stockholm, Sweden. **Dept of Lung Medicine, Huddinge University Hospital, Huddinge, Sweden. † Central Microbiological Laboratory, Stockholm County Council, Stockholm, Sweden. Correspondence: S.E. Hoffner Dept of Bacteriology National Bacteriological Laboratory S-105 21 Stockholm Sweden Keywords: Combined drug therapy drug resistance drug synergism Mycobacterium avium complex Received: February 19 1993 Accepted after revision August 15 1993 ethambutol/rifampicin and ethambutol/streptomycin, of MAC strains isolated in the early and late phase of disease from patients on long-term drug therapy due to severe MAC infections. Methods Patients and isolates Initial and later isolates of MAC from five patients who had been on long-term (3–5 yrs) treatment for MAC infections (table 1) were studied. All strains were isolated by culture on Löwenstein-Jensen egg medium, or with the Bactec broth system (Becton & Dickinson, Md, USA). The isolates were identified to species level at the National Bacteriological Laboratory, by conventional reference techniques [11]. The patients were included in the study if they showed a clinical and bacteriological response, in spite of prolonged treatment. All patients had repeated isolates of MAC [5–14], and fulfilled the criteria of disease with atypical mycobacteria listed by the American Thoracic Society [12]. Three of the patients were adults with pulmonary disease, one had a disseminated infection after a kidney transplantation (with isolates from biopsies, fistula and sputum), and one was a HIV-negative boy with disseminated MAC infection. Three patients died within one year after the last isolation. The intervals between isolation of the early and late strains studied were 3–5 yrs. 10 62 51 73 68 1 2 3 4 5 F F F M M Sex M/F Status post-tuberculosis Bronchiectases Renal transplant prednisolone azathioprine Gastric resection, pernicicous anaemia Uncharacterized cellular immunodeficiency Predisposing factors Dyspnoea, fever, cough Fever, cough Large bilateral infiltrates Progress of infiltrates Fever spikes BAL fluid (b) Sputum (a) Sputum (b) Sputum (a) Urine (b) Large infiltrates Large bilateral infiltrates Urine (a) Sputum (b) Infilatrates right Bilateral large infiltrates Fine nodular infiltrates Sputum (a) Blood (b) Gastric washing (a) Susceptibility of early (a) and late (b) MAC isolates Large infiltrates left Fever, cough Tumours* both breasts Fever, cough High fever, Uraemia High fever Fever, weight loss Normal Abdominal abscess Fever spikes Normal Normal Lung X-ray Fever, weight loss, anaemia Symptoms 1 yr IN, EB, AM 1 yr 0 2 months SM, RB, EB 16 months RB, EB 6 months 0 2 months RB, EB 1 yr 0 3 months SM, EB, RIF 4 months 0 2 yrs RB, AM, EB 1 yr RB, EB 6 months 0 1 yr RIF, EB, AM 3 yrs 0 3 weeks RB, EB, AM 4 months IN 18 months RB, EB, AM 2 yrs IN, EB, RIF 6 months 0 18 months RB, EB, AM 3 weeks 0 4 days RB, EB, AM 2 yrs IN, RIF, SM, EB 1 yr 0 2 yrs, RB, EB, AM, Cy 1 yr, RB, EB, La, Cy, CM 1 yr RB, EB, La, Cy, CM Therapy Death Relapse Chronic pulmonary disease Relapse Relapse Death Death Relapse Lobectomy left lung Chronic disseminated disease Relapse Course *: Patho-anatomical diagnosis of biopsy; -: Langhans' giant cells and caseous necrosis, culture not performed. AM: amikacin; CM: clarithromycin; Cy: cycloserine; EB: ethambutol; IN: isoniazid; La: Lamprene (clofazimine); PZ: pyrazinamide; RIF: rifampicin; RB: rifabutin; MAC: Mycobacterium avium complex; BAL: bronchoalveolar lavage; Pt: patient; M: male; F: female. Age at initial presentation yrs Pt No. Table 1. – Patients with chronic MAC infections in long-term drug therapy 248 S.E. HOFFNER ET AL. M . AV I U M D E V E L O P R E S I S TA N C E TO C O M B I N E D D RU G S The drugs used for susceptibility testing were obtained as powders: ethambutol (EB), (Cyanamid, UK); rifampicin (RIF) (Ferrosan, Sweden), and streptomycin (SM), (Glaxo Laboratories, UK). A stock solution of each drug was prepared in sterile 0.067 M phosphate buffer (PBS) at pH 7.2, at a concentration 40 times the test concentration used. RIF was dissolved in a small amount of dimethylsulphoxide before PBS was added. To each test vial containing 4 ml of culturing medium, 0.1 ml of the stock solution was added, to give the following testconcentrations; EB 5.0 mg·l-1, RIF 2.0 mg·l-1, and SM 4.0 mg·l-1. These concentrations were established after studies of the inhibitory effects on MAC of serial dilutions of each drug, and taking the achievable serum concentrations into account. Susceptibility testing The Bactec radiometric system (Becton & Dickinson, Md, USA) and the 7H12B Middlebrook TB medium, an enriched 7H9 broth supplemented with bovine serum albumin, catalase, casein hydrolysate and 14C-labelled palmitic acid [13], were used for the susceptibility testing. The tested MAC strains were subcultured on Löwenstein-Jensen egg medium at 37°C for 3–4 weeks, before being suspended in PBS to a bacterial density corresponding to McFarland 0.5 standard. After homogenization, this suspension was further diluted 1/10 in PBS, and 0.1 ml was inoculated into Bactec culturing-vials containing a drug or drug-combination, and to one drugfree control vial, giving a final concentration in the culturing-vials of approximately 1×105 colony forming units (CFU)·ml-1 [5]. In a second control vial, a 1/100 dilution of the bacterial suspension was used as inoculum. The two MAC isolates from each patient were examined in parallel. The 14CO2 produced by metabolically active mycobacteria was quantified daily in a Bactec 460 instrument (Becton & Dickinson, Md, USA), over a 4 day period. The results were expressed as growth index (GI), values ranging from 0–999. The principles for the evaluation of drug interactions and for determining mycobacterial drug susceptibility to combined drugs have been reported previously [5, 7]. In short, the interpretation of the test is based on the amount and kinetics of the growth/growth-inhibition of a drug-exposed culture, compared to the drug-free control cultures. A drug-exposed isolate was defined as susceptible to the tested drug/drugcombination when the radiometric GI value on day 4 was decreasing, thus revealing an increased inhibition of the metabolic activity of the cultured mycobacteria. Table 2. – Susceptibility to various drugs/drug combinations of early and late isolates from patients with longterm MAC infection Pt No. Isolate EB RIF SM EB+RIF EB+SM 1 a) 1984* b) 1989 R R R R R R S R S R 2 a) 1985 b) 1988 R R R R R I S R S I 3 a) 1984 b) 1989 R R R R R R S R S R 4 a) 1985 b) 1989 R I R R R R I R S S 5 a) 1986 b) 1989 I R R R R R S R S S *: year of isolation. MAC: Mycobacterium avium complex; EB: ethambutol; I: intermediate; R: resistant; RIF: rifampicin; S: susceptible; SM: streptomycin. EB/SM, and all but one EB/RIF also (table 2 and fig. 1). All five late isolates were resistant to the combination of EB/RIF, and three strains had also developed resistance to EB/SM (table 2). During the interval between the isolation of the respective pairs of isolates, all patients had been treated with EB/RIF or EB/rifabutin (RB) for at least 18 months. The patients whose strains had developed resistance to the combination of EB/SM had been on treatment for 2 yrs of SM (patient No. 1), or 18 months of amikacin (AM) (patients Nos 2 and 3), between the two occasions of isolation. 1200 1000 800 Growth index Drugs 600 400 200 0 0 Results All isolates were resistant or moderately resistant to the drugs tested singly; EB (5.0 mg·l-1), RIF (2.0 mg·l-1) and SM (4.0 mg·l-1). The early isolates from each of the five patients were susceptible to the combination of 249 2 4 6 8 Day Fig. 1. – Growth kinetics for early and late MAC isolates from patient No. 1 exposed to the drug-combinations of ethambutol, 5 mg·l-1 (EB) with streptomycin, 4 mg·l-1 (SM) or rifampicin 2 mg·l-1 (RIF), as reflected by Bactec growth index values. : 1984, SM+EB; : 1989, SM+EB; : 1984, RIF+EB; : 1989, RIF+EB. MAC: Mycobacterium avium complex. S.E. HOFFNER ET AL. 250 Discussion Currently, there is a propensity to regard MAC strains as resistant to most anti-mycobacterial drugs and, consequently, treatment failures are often explained by this inherent drug resistance. However, there are good clinical and laboratory data showing that combination therapy is effective in many cases. Synergistic drug interactions seem to be of central importance for treatment of MAC infections [14], and ethambutol seems to be a key-drug in achieving such anti-mycobacterial drug synergy [15]. Combinations of EB with RIF or SM also act synergistically on most MAC isolates in vitro [5]. Hence, in most cases, strains of MAC are highly susceptible to these drug-combinations. In this study, we isolated MAC strains from five patients during the course of long-term MAC infection, and determined the susceptibility of the strains to some anti-mycobacterial drugs and their combinations. Between the isolation of the early and late MAC isolates, all patients had been on treatment with EB in combination with a rifamycin (RIF or RB) and an aminoglycoside (SM or AM) for at least 18 months. While all of the isolates collected in the early phase of disease were susceptible to both drug combinations, all isolates collected late in the course of disease showed resistance to the combination of EB/RIF, and most of them also to the EB/SM combination. Hence, treatment of patients with initially effective synergistically acting combinations of EB with either a rifamycin or an aminoglycoside, may lead to in vivo selection of resistant escape variants of MAC to both of these drug-combination. One may hypothesize that the resistant strains are new strains causing reinfection. However, since initial resistance to combinations of drugs is rare [5], the most probable cause is reactivation with the same strain. Also, in some instances, isolates in between early and late strains were studied, and intermediate resistance against combinations of drugs was observed (data not shown), supporting the concept that resistance developed during therapy. Increased drug resistance of MAC during therapy, e.g. to rifampicin in one out of seven patients tested, has been reported previously [16], but only to certain drugs when tested separately. 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