AAC Accepted Manuscript Posted Online 16 February 2016 Antimicrob. Agents Chemother. doi:10.1128/AAC.02864-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1
Tigecycline potentiates clarithromycin activity against Mycobacterium avium in
2
vitro
3 4
Hannelore I. Baxa#, Irma A.J.M. Bakker-Woudenbergb, Marian T. ten Kateb, Annelies
5
Verbona,b and Jurriaan E.M. de Steenwinkelb
6 7 8
Department of Internal Medicine, Section of Infectious Diseases, Erasmus University
9
Medical Centre, Rotterdam, the Netherlandsa; Department of Medical Microbiology
10
and Infectious Diseases, Erasmus University Medical Centre, Rotterdam, the
11
Netherlandsb
12 13
Running head: tigecycline activity against M. avium
14 15 16 17 18 19
# Address correspondence to Hannelore I. Bax, [email protected]
20 21 22 23 24
1
25
Abstract
26 27
The in vitro activity of clarithromycin and tigecycline alone and in combination against
28
Mycobacterium avium was assessed. The activity of clarithromycin was time-
29
dependent, highly variable and often resulted in clarithromycin resistance.
30
Tigecycline showed concentration-dependent activity and mycobacterial killing could
31
only be achieved at high concentrations. Tigecycline enhanced clarithromycin activity
32
against M. avium and prevented clarithromycin resistance. Whether there is clinical
33
usefulness of tigecycline in the treatment of M. avium infections needs further study.
34 35 36 37
2
38
Although the introduction of macrolides improved treatment success of
39
Mycobacterium avium infections, overall prognosis is still poor (1). Therefore, new
40
powerful treatment strategies are needed. Tigecycline has been shown to have good
41
in vitro activity against rapidly growing non-tuberculous mycobacteria (NTM) (2-4)
42
and success in clinical use has been reported when combined with macrolides (5, 6).
43
In contrast, little is known about tigecycline activity against slowly growing NTM
44
including M. avium and in vitro activity has not been demonstrated so far (7). In the
45
present study, the bactericidal activity of clarithromycin and tigecycline alone and in
46
combination against M. avium was assessed.
47
Suspensions of M. avium complex (MAC) 101 strain (kindly provided by L.S.
48
Young, Kuzell Institute for Arthritis and Infectious Diseases, USA) were cultured in
49
Middlebrook 7H9 broth (Difco Laboratories, USA), supplemented with 10% oleic acid-
50
albumin-dextrose-catalase (OADC; Baltimore Biological Laboratories, USA), 0.5%
51
glycerol (Scharlau Chemie SA, Spain) and 0.02% Tween 20 (Sigma Chemical Co.,
52
USA) under shaking conditions at 96 rpm at 37ºC. Cultures on solid medium were
53
grown on Middlebrook 7H10 agar (Difco), supplemented with 10% OADC and 0.5%
54
glycerol for 14 days at 37ºC with 5% CO2. The susceptibility of the M. avium strain in
55
terms of MICs determined according to the guidelines of the Clinical and Laboratory
56
Standards Institutes (CLSI) (8) were 2 mg/L for clarithromycin and 16 mg/L for
57
tigecycline. The concentration- and time-dependent killing capacity of clarithromycin
58
and tigecycline was determined as previously described (9, 10). Briefly, M. avium
59
cultures were exposed to antimicrobial drugs at 4-fold increasing concentrations for
60
21 days at 37°C under shaking conditions. At day 1, 3, 7, 10 and 21 during exposure,
61
samples were collected, centrifuged at 14000xg and subcultured onto antibiotic-free
62
solid medium. Subculture plates were incubated for 14 days at 37°C with 5% CO2 to
3
63
determine the number of colony forming units (cfu). The lower limit of quantification
64
was 5 cfu/mL (log 0.7). To assess selection of clarithromycin-resistant M. avium,
65
subcultures were also performed on clarithromycin-containing (32 mg/L) solid
66
medium. The stability of clarithromycin and tigecycline in mycobacteria-free
67
Middlebrook 7H9 broth at 37 °C was determined using two test concentrations of
68
clarithromycin and tigecycline (8 and 32 mg/L). Antimicrobial activity over time was
69
assessed using the standard large-plate agar diffusion assay as previously described
70
(11). In contrast to clarithromycin (showing no decline during 21 days of exposure),
71
tigecycline concentrations fell to 20% of the original concentrations within the first 24
72
hours and thus 80% of the original tigecycline concentrations was added daily. The
73
two endpoints of this study were drug synergy and the prevention of the emergence
74
of clarithromycin-resistance. Synergistic activity was defined as a ≥ 100-fold (2log10)
75
increase in mycobacterial killing with the combination compared to the most active
76
single drug or when the drug combination achieved elimination of M. avium after 21
77
days of drug exposure which was not achieved during single drug exposure (12, 13).
78
Clarithromycin showed time-dependent bactericidal activity towards M. avium
79
(figure 1a). At the intermediate concentrations (2 and 8 mg/L) high inter-experimental
80
variability was observed showing mycobacterial killing in 2 out of 4 experiments and
81
mycobacterial regrowth in the other 2 experiments, which was associated with the
82
selection of clarithromycin resistance. Only at the highest concentration tested (32
83
mg/L) ≥99% killing was consistently observed. Tigecycline showed concentration-
84
dependent bactericidal activity towards M. avium (figure 1b). At tigecycline
85
concentrations of ≥ 8 mg/L, ≥99% killing was observed and mycobacterial elimination
86
was achieved only at the highest concentration tested (32 mg/L).
4
87
A concentration-dependent enhancement of clarithromycin activity by tigecycline was
88
observed (figure 2a, 2b, table 1). Whereas low concentration tigecycline (0.125 mg/L)
89
effected synergy in combination with clarithromycin at ≥ 2 mg/L, tigecycline at 0.5
90
mg/L effected synergy with clarithromycin at ≥ 0.5 mg/L and tigecycline at 2 mg/L
91
effected synergy with clarithromycin at ≥ 0.125 mg/L, except with clarithromycin 2
92
mg/L. All tested tigecycline concentrations prevented the emergence of
93
clarithromycin resistance when combined with clarithromycin at 0.125 – 8 mg/L.
94
To our knowledge, this is the first study showing that the addition of tigecycline
95
to clarithromycin increased bactericidal activity against M. avium and prevented the
96
selection of clarithromycin resistance. Recently, Huang et al. showed that tigecycline
97
could potentiate clarithromycin activity against rapidly growing mycobacteria (RGM)
98
in vitro (2) supporting the use of this combination in the treatment of infections
99
caused by RGM. In humans, the steady-state maximum serum concentration at
100
clinical dosages of tigecycline is around 0.6 mg/L (14). Although this concentration is
101
far below the tigecycline concentrations needed to achieve mycobacterial killing in
102
our in vitro study, it approximates the concentrations effecting synergy in combination
103
with clarithromycin in our study. Moreover, tigecycline has been shown to accumulate
104
in tissues and human macrophages (14, 15). Further studies including macrophage-
105
infection and pharmacodynamic/pharmacokinetic models, and in vivo models are
106
needed to establish to what extent tigecycline can contribute to killing and elimination
107
of M. avium and whether tigecycline can indeed effect synergy in combination with
108
clarithromycin as we have shown in the present study. Although clarithromycin is
109
considered the cornerstone agent in the treatment of M. avium infections, consistent
110
mycobacterial killing was only seen at the highest concentration tested (32 mg/L).
111
Importantly, at clinically relevant concentrations clarithromycin activity against M.
5
112
avium was highly variable between experiments alternating mycobacterial killing and
113
mycobacterial regrowth. These results might explain the fact that despite macrolide-
114
containing regimens, overall treatment success of M. avium infections is still
115
unpredictable and disappointing (1). The results of our study also illustrate that the
116
dynamic time-kill kinetics assay can detect important differences in antibacterial drug
117
behaviour that cannot be detected when using static susceptibility assays such as
118
MICs. This is in line with our previous studies (10, 16) as well as with another recent
119
study assessing the activity of several antibacterial drugs against Mycobacterium
120
abscessus (17). The results of our in vitro study underline the need for potentiating
121
clarithromycin activity against M. avium. Whether there might be clinical usefulness of
122
tigecycline when added to clarithromycin based regimens needs further study.
123 124 125
Funding Information
126
This research received no specific grant from any funding agency in the public,
127
commercial, or not-for-profit sectors
128
6
129
Literature
130
1.
van Ingen J, Ferro BE, Hoefsloot W, Boeree MJ, van Soolingen D. 2013.
131
Drug treatment of pulmonary nontuberculous mycobacterial disease in HIV-
132
negative patients: the evidence. Expert Rev Anti Infect Ther 11:1065-1077.
133
2.
Huang CW, Chen JH, Hu ST, Huang WC, Lee YC, Huang CC, Shen GH.
134
2013. Synergistic activities of tigecycline with clarithromycin or amikacin
135
against rapidly growing mycobacteria in Taiwan. Int J Antimicrob Agents
136
41:218-223.
137
3.
Lee MR, Ko JC, Liang SK, Lee SW, Yen DH, Hsueh PR. 2014. Bacteraemia
138
caused by Mycobacterium abscessus subsp. abscessus and M. abscessus
139
subsp. bolletii: clinical features and susceptibilities of the isolates. Int J
140
Antimicrob Agents 43:438-441.
141
4.
Singh S, Bouzinbi N, Chaturvedi V, Godreuil S, Kremer L. 2014. In vitro
142
evaluation of a new drug combination against clinical isolates belonging to the
143
Mycobacterium abscessus complex. Clin Microbiol Infect doi:10.1111/1469-
144
0691.12780.
145
5.
Garrison AP, Morris MI, Doblecki Lewis S, Smith L, Cleary TJ, Procop
146
GW, Vincek V, Rosa-Cunha I, Alfonso B, Burke GW, Tzakis A, Hartstein
147
AI. 2009. Mycobacterium abscessus infection in solid organ transplant
148
recipients: report of three cases and review of the literature. Transpl Infect Dis
149
11:541-548.
150
6.
Wallace RJ, Jr., Dukart G, Brown-Elliott BA, Griffith DE, Scerpella EG,
151
Marshall B. 2014. Clinical experience in 52 patients with tigecycline-
152
containing regimens for salvage treatment of Mycobacterium abscessus and
153
Mycobacterium chelonae infections. J Antimicrob Chemother 69:1945-1953.
7
154
7.
Wallace RJ, Jr., Brown-Elliott BA, Crist CJ, Mann L, Wilson RW. 2002.
155
Comparison of the in vitro activity of the glycylcycline tigecycline (formerly
156
GAR-936) with those of tetracycline, minocycline, and doxycycline against
157
isolates of nontuberculous mycobacteria. Antimicrob Agents Chemother
158
46:3164-3167.
159
8.
Clinical and Laboratory Standards Institute. 2011. Susceptibility Testing of
160
Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes; Approved
161
Standard-Second Edition. CLSI document M24-A2 Wayne, PA, USA.
162
9.
Bakker-Woudenberg IA, van Vianen W, van Soolingen D, Verbrugh HA,
163
van Agtmael MA. 2005. Antimycobacterial agents differ with respect to their
164
bacteriostatic versus bactericidal activities in relation to time of exposure,
165
mycobacterial growth phase, and their use in combination. Antimicrob Agents
166
Chemother 49:2387-2398.
167
10.
de Steenwinkel JE, de Knegt GJ, ten Kate MT, van Belkum A, Verbrugh
168
HA, Kremer K, van Soolingen D, Bakker-Woudenberg IA. 2010. Time-kill
169
kinetics of anti-tuberculosis drugs, and emergence of resistance, in relation to
170
metabolic activity of Mycobacterium tuberculosis. J Antimicrob Chemother
171
65:2582-2589.
172
11.
method for antibiotic assay of clinical specimens. Appl Microbiol 14:170-177.
173 174
Bennett JV, Brodie JL, Benner EJ, Kirby WM. 1966. Simplified, accurate
12.
Eliopoulos GM, Moellering Jr RC. 1991. Antimicrobial Combinations, p 432-
175
480. In Loran V (ed), Antibiotics in Laboratory Medicine, 3rd ed, Baltimore,
176
Maryland, USA.
177 178
13.
Doern CD. 2014. When does 2 plus 2 equal 5? A review of antimicrobial synergy testing. J Clin Microbiol 52:4124-4128.
8
179
14.
Meagher AK, Ambrose PG, Grasela TH, Ellis-Grosse EJ. 2005. The
180
pharmacokinetic and pharmacodynamic profile of tigecycline. Clin Infect Dis
181
41 Suppl 5:S333-340.
182
15.
Bopp LH, Baltch AL, Ritz WJ, Michelsen PB, Smith RP. 2011. Activities of
183
tigecycline and comparators against Legionella pneumophila and Legionella
184
micdadei extracellularly and in human monocyte-derived macrophages. Diagn
185
Microbiol Infect Dis 69:86-93.
186
16.
de Steenwinkel JEM, ten Kate MT, de Knegt GJ, Kremer K, Aarnoutse RE,
187
Boeree MJ, van Soolingen D, Bakker-Woudenberg IA. 2012. Drug
188
susceptibility of Mycobacterium tuberculosis Beijing genotype and association
189
with MDR TB. Emerg Infect Dis 18:660-663.
190
17.
Ferro BE, van Ingen J, Wattenberg M, van Soolingen D, Mouton JW.
191
2015. Time-kill kinetics of antibiotics active against rapidly growing
192
mycobacteria. J Antimicrob Chemother 70:811-817.
193 194 195 196 197
9
CLR 0 CLR 0.125 CLR 0.5 CLR 2 CLR 8
TGC 0 CLR-Res 1:4.5E7* 0.31% 0.06% 80% 54%
TGC 0.125 Synergy CLR-Res + +(E)
1:1.2E7 1:1.0E7 nd 0
TGC 0.5 Synergy CLR-Res + +(E) +
1:1.5E7 0 0 0
TGC 2 Synergy CLR-Res + + +
0 0 0 0
198 199
Table 1. Synergistic activity (Synergy) and prevention of selection of clarithromycin resistance (CLR-Res) in M. avium after 21 days
200
of drug exposure. CLR clarithromycin, TGC tigecycline, *spontaneous mutation frequency, + synergy, E elimination (< 5 cfu/mL =
201
limit of quantification), nd not determined.
202
10
203
Figure legends
204 205
Figure 1a. Concentration- and time-dependent bactericidal activity of clarithromycin
206
towards M. avium.
207 208
Figure 1b. Concentration- and time-dependent bactericidal activity of tigecycline
209
towards M. avium.
210 211
Figure 2a. Concentration- and time-dependent bactericidal activity of clarithromycin 2
212
mg/L combined with tigecycline at various concentrations towards M. avium.
213 214
Figure 2b. Concentration- and time-dependent bactericidal activity of clarithromycin
215
8 mg/L combined with tigecycline at various concentrations towards M. avium.
216 217 218 219 220 221 222 223 224
11
Figure 1a. Concentration- and time-dependent bactericidal activity of clarithromycin towards M. avium.
Figure 1b. Concentration- and time-dependent bactericidal activity of tigecycline towards M. avium.
Figure 2a. Concentration- and time-dependent bactericidal activity of clarithromycin 2 mg/L combined with tigecycline at various concentrations towards M. avium.
Figure 2b. Concentration- and time-dependent bactericidal activity of clarithromycin 8 mg/L combined with tigecycline at various concentrations towards M. avium.
1
Tigecycline potentiates clarithromycin activity against Mycobacterium avium in
2
vitro
3 4
Hannelore I. Baxa#, Irma A.J.M. Bakker-Woudenbergb, Marian T. ten Kateb, Annelies
5
Verbona,b and Jurriaan E.M. de Steenwinkelb
6 7 8
Department of Internal Medicine, Section of Infectious Diseases, Erasmus University
9
Medical Centre, Rotterdam, the Netherlandsa; Department of Medical Microbiology
10
and Infectious Diseases, Erasmus University Medical Centre, Rotterdam, the
11
Netherlandsb
12 13
Running head: tigecycline activity against M. avium
14 15 16 17 18 19
# Address correspondence to Hannelore I. Bax, [email protected]
20 21 22 23 24
1
25
Abstract
26 27
The in vitro activity of clarithromycin and tigecycline alone and in combination against
28
Mycobacterium avium was assessed. The activity of clarithromycin was time-
29
dependent, highly variable and often resulted in clarithromycin resistance.
30
Tigecycline showed concentration-dependent activity and mycobacterial killing could
31
only be achieved at high concentrations. Tigecycline enhanced clarithromycin activity
32
against M. avium and prevented clarithromycin resistance. Whether there is clinical
33
usefulness of tigecycline in the treatment of M. avium infections needs further study.
34 35 36 37
2
38
Although the introduction of macrolides improved treatment success of
39
Mycobacterium avium infections, overall prognosis is still poor (1). Therefore, new
40
powerful treatment strategies are needed. Tigecycline has been shown to have good
41
in vitro activity against rapidly growing non-tuberculous mycobacteria (NTM) (2-4)
42
and success in clinical use has been reported when combined with macrolides (5, 6).
43
In contrast, little is known about tigecycline activity against slowly growing NTM
44
including M. avium and in vitro activity has not been demonstrated so far (7). In the
45
present study, the bactericidal activity of clarithromycin and tigecycline alone and in
46
combination against M. avium was assessed.
47
Suspensions of M. avium complex (MAC) 101 strain (kindly provided by L.S.
48
Young, Kuzell Institute for Arthritis and Infectious Diseases, USA) were cultured in
49
Middlebrook 7H9 broth (Difco Laboratories, USA), supplemented with 10% oleic acid-
50
albumin-dextrose-catalase (OADC; Baltimore Biological Laboratories, USA), 0.5%
51
glycerol (Scharlau Chemie SA, Spain) and 0.02% Tween 20 (Sigma Chemical Co.,
52
USA) under shaking conditions at 96 rpm at 37ºC. Cultures on solid medium were
53
grown on Middlebrook 7H10 agar (Difco), supplemented with 10% OADC and 0.5%
54
glycerol for 14 days at 37ºC with 5% CO2. The susceptibility of the M. avium strain in
55
terms of MICs determined according to the guidelines of the Clinical and Laboratory
56
Standards Institutes (CLSI) (8) were 2 mg/L for clarithromycin and 16 mg/L for
57
tigecycline. The concentration- and time-dependent killing capacity of clarithromycin
58
and tigecycline was determined as previously described (9, 10). Briefly, M. avium
59
cultures were exposed to antimicrobial drugs at 4-fold increasing concentrations for
60
21 days at 37°C under shaking conditions. At day 1, 3, 7, 10 and 21 during exposure,
61
samples were collected, centrifuged at 14000xg and subcultured onto antibiotic-free
62
solid medium. Subculture plates were incubated for 14 days at 37°C with 5% CO2 to
3
63
determine the number of colony forming units (cfu). The lower limit of quantification
64
was 5 cfu/mL (log 0.7). To assess selection of clarithromycin-resistant M. avium,
65
subcultures were also performed on clarithromycin-containing (32 mg/L) solid
66
medium. The stability of clarithromycin and tigecycline in mycobacteria-free
67
Middlebrook 7H9 broth at 37 °C was determined using two test concentrations of
68
clarithromycin and tigecycline (8 and 32 mg/L). Antimicrobial activity over time was
69
assessed using the standard large-plate agar diffusion assay as previously described
70
(11). In contrast to clarithromycin (showing no decline during 21 days of exposure),
71
tigecycline concentrations fell to 20% of the original concentrations within the first 24
72
hours and thus 80% of the original tigecycline concentrations was added daily. The
73
two endpoints of this study were drug synergy and the prevention of the emergence
74
of clarithromycin-resistance. Synergistic activity was defined as a ≥ 100-fold (2log10)
75
increase in mycobacterial killing with the combination compared to the most active
76
single drug or when the drug combination achieved elimination of M. avium after 21
77
days of drug exposure which was not achieved during single drug exposure (12, 13).
78
Clarithromycin showed time-dependent bactericidal activity towards M. avium
79
(figure 1a). At the intermediate concentrations (2 and 8 mg/L) high inter-experimental
80
variability was observed showing mycobacterial killing in 2 out of 4 experiments and
81
mycobacterial regrowth in the other 2 experiments, which was associated with the
82
selection of clarithromycin resistance. Only at the highest concentration tested (32
83
mg/L) ≥99% killing was consistently observed. Tigecycline showed concentration-
84
dependent bactericidal activity towards M. avium (figure 1b). At tigecycline
85
concentrations of ≥ 8 mg/L, ≥99% killing was observed and mycobacterial elimination
86
was achieved only at the highest concentration tested (32 mg/L).
4
87
A concentration-dependent enhancement of clarithromycin activity by tigecycline was
88
observed (figure 2a, 2b, table 1). Whereas low concentration tigecycline (0.125 mg/L)
89
effected synergy in combination with clarithromycin at ≥ 2 mg/L, tigecycline at 0.5
90
mg/L effected synergy with clarithromycin at ≥ 0.5 mg/L and tigecycline at 2 mg/L
91
effected synergy with clarithromycin at ≥ 0.125 mg/L, except with clarithromycin 2
92
mg/L. All tested tigecycline concentrations prevented the emergence of
93
clarithromycin resistance when combined with clarithromycin at 0.125 – 8 mg/L.
94
To our knowledge, this is the first study showing that the addition of tigecycline
95
to clarithromycin increased bactericidal activity against M. avium and prevented the
96
selection of clarithromycin resistance. Recently, Huang et al. showed that tigecycline
97
could potentiate clarithromycin activity against rapidly growing mycobacteria (RGM)
98
in vitro (2) supporting the use of this combination in the treatment of infections
99
caused by RGM. In humans, the steady-state maximum serum concentration at
100
clinical dosages of tigecycline is around 0.6 mg/L (14). Although this concentration is
101
far below the tigecycline concentrations needed to achieve mycobacterial killing in
102
our in vitro study, it approximates the concentrations effecting synergy in combination
103
with clarithromycin in our study. Moreover, tigecycline has been shown to accumulate
104
in tissues and human macrophages (14, 15). Further studies including macrophage-
105
infection and pharmacodynamic/pharmacokinetic models, and in vivo models are
106
needed to establish to what extent tigecycline can contribute to killing and elimination
107
of M. avium and whether tigecycline can indeed effect synergy in combination with
108
clarithromycin as we have shown in the present study. Although clarithromycin is
109
considered the cornerstone agent in the treatment of M. avium infections, consistent
110
mycobacterial killing was only seen at the highest concentration tested (32 mg/L).
111
Importantly, at clinically relevant concentrations clarithromycin activity against M.
5
112
avium was highly variable between experiments alternating mycobacterial killing and
113
mycobacterial regrowth. These results might explain the fact that despite macrolide-
114
containing regimens, overall treatment success of M. avium infections is still
115
unpredictable and disappointing (1). The results of our study also illustrate that the
116
dynamic time-kill kinetics assay can detect important differences in antibacterial drug
117
behaviour that cannot be detected when using static susceptibility assays such as
118
MICs. This is in line with our previous studies (10, 16) as well as with another recent
119
study assessing the activity of several antibacterial drugs against Mycobacterium
120
abscessus (17). The results of our in vitro study underline the need for potentiating
121
clarithromycin activity against M. avium. Whether there might be clinical usefulness of
122
tigecycline when added to clarithromycin based regimens needs further study.
123 124 125
Funding Information
126
This research received no specific grant from any funding agency in the public,
127
commercial, or not-for-profit sectors
128
6
129
Literature
130
1.
van Ingen J, Ferro BE, Hoefsloot W, Boeree MJ, van Soolingen D. 2013.
131
Drug treatment of pulmonary nontuberculous mycobacterial disease in HIV-
132
negative patients: the evidence. Expert Rev Anti Infect Ther 11:1065-1077.
133
2.
Huang CW, Chen JH, Hu ST, Huang WC, Lee YC, Huang CC, Shen GH.
134
2013. Synergistic activities of tigecycline with clarithromycin or amikacin
135
against rapidly growing mycobacteria in Taiwan. Int J Antimicrob Agents
136
41:218-223.
137
3.
Lee MR, Ko JC, Liang SK, Lee SW, Yen DH, Hsueh PR. 2014. Bacteraemia
138
caused by Mycobacterium abscessus subsp. abscessus and M. abscessus
139
subsp. bolletii: clinical features and susceptibilities of the isolates. Int J
140
Antimicrob Agents 43:438-441.
141
4.
Singh S, Bouzinbi N, Chaturvedi V, Godreuil S, Kremer L. 2014. In vitro
142
evaluation of a new drug combination against clinical isolates belonging to the
143
Mycobacterium abscessus complex. Clin Microbiol Infect doi:10.1111/1469-
144
0691.12780.
145
5.
Garrison AP, Morris MI, Doblecki Lewis S, Smith L, Cleary TJ, Procop
146
GW, Vincek V, Rosa-Cunha I, Alfonso B, Burke GW, Tzakis A, Hartstein
147
AI. 2009. Mycobacterium abscessus infection in solid organ transplant
148
recipients: report of three cases and review of the literature. Transpl Infect Dis
149
11:541-548.
150
6.
Wallace RJ, Jr., Dukart G, Brown-Elliott BA, Griffith DE, Scerpella EG,
151
Marshall B. 2014. Clinical experience in 52 patients with tigecycline-
152
containing regimens for salvage treatment of Mycobacterium abscessus and
153
Mycobacterium chelonae infections. J Antimicrob Chemother 69:1945-1953.
7
154
7.
Wallace RJ, Jr., Brown-Elliott BA, Crist CJ, Mann L, Wilson RW. 2002.
155
Comparison of the in vitro activity of the glycylcycline tigecycline (formerly
156
GAR-936) with those of tetracycline, minocycline, and doxycycline against
157
isolates of nontuberculous mycobacteria. Antimicrob Agents Chemother
158
46:3164-3167.
159
8.
Clinical and Laboratory Standards Institute. 2011. Susceptibility Testing of
160
Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes; Approved
161
Standard-Second Edition. CLSI document M24-A2 Wayne, PA, USA.
162
9.
Bakker-Woudenberg IA, van Vianen W, van Soolingen D, Verbrugh HA,
163
van Agtmael MA. 2005. Antimycobacterial agents differ with respect to their
164
bacteriostatic versus bactericidal activities in relation to time of exposure,
165
mycobacterial growth phase, and their use in combination. Antimicrob Agents
166
Chemother 49:2387-2398.
167
10.
de Steenwinkel JE, de Knegt GJ, ten Kate MT, van Belkum A, Verbrugh
168
HA, Kremer K, van Soolingen D, Bakker-Woudenberg IA. 2010. Time-kill
169
kinetics of anti-tuberculosis drugs, and emergence of resistance, in relation to
170
metabolic activity of Mycobacterium tuberculosis. J Antimicrob Chemother
171
65:2582-2589.
172
11.
method for antibiotic assay of clinical specimens. Appl Microbiol 14:170-177.
173 174
Bennett JV, Brodie JL, Benner EJ, Kirby WM. 1966. Simplified, accurate
12.
Eliopoulos GM, Moellering Jr RC. 1991. Antimicrobial Combinations, p 432-
175
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193 194 195 196 197
9
CLR 0 CLR 0.125 CLR 0.5 CLR 2 CLR 8
TGC 0 CLR-Res 1:4.5E7* 0.31% 0.06% 80% 54%
TGC 0.125 Synergy CLR-Res + +(E)
1:1.2E7 1:1.0E7 nd 0
TGC 0.5 Synergy CLR-Res + +(E) +
1:1.5E7 0 0 0
TGC 2 Synergy CLR-Res + + +
0 0 0 0
198 199
Table 1. Synergistic activity (Synergy) and prevention of selection of clarithromycin resistance (CLR-Res) in M. avium after 21 days
200
of drug exposure. CLR clarithromycin, TGC tigecycline, *spontaneous mutation frequency, + synergy, E elimination (< 5 cfu/mL =
201
limit of quantification), nd not determined.
202
10
203
Figure legends
204 205
Figure 1a. Concentration- and time-dependent bactericidal activity of clarithromycin
206
towards M. avium.
207 208
Figure 1b. Concentration- and time-dependent bactericidal activity of tigecycline
209
towards M. avium.
210 211
Figure 2a. Concentration- and time-dependent bactericidal activity of clarithromycin 2
212
mg/L combined with tigecycline at various concentrations towards M. avium.
213 214
Figure 2b. Concentration- and time-dependent bactericidal activity of clarithromycin
215
8 mg/L combined with tigecycline at various concentrations towards M. avium.
216 217 218 219 220 221 222 223 224
11
Figure 1a. Concentration- and time-dependent bactericidal activity of clarithromycin towards M. avium.
Figure 1b. Concentration- and time-dependent bactericidal activity of tigecycline towards M. avium.
Figure 2a. Concentration- and time-dependent bactericidal activity of clarithromycin 2 mg/L combined with tigecycline at various concentrations towards M. avium.
Figure 2b. Concentration- and time-dependent bactericidal activity of clarithromycin 8 mg/L combined with tigecycline at various concentrations towards M. avium.
