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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. This is the first report on the formation of MAC strains resistant to synergistically active
drug-combinations during long-term drug therapy. These
findings emphasize the need for careful in vitro monitoring of drug resistance to combined drugs, to enable
rapid detection of such highly resistant MAC strains, and
prompt modification of the drug regimen, thereafter.
Acknowledgements: Financial support by King Oscar
II:s Jubilee Foundation is gratefully acknowledged.
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