International Journal of Antimicrobial Agents 45 (2015) 71–75
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short Communication
In vitro synergistic activity of clofazimine and other antituberculous drugs against multidrug-resistant Mycobacterium tuberculosis isolates Zhijian Zhang a,b,1 , Tianzhi Li a,1 , Geping Qu a , Yu Pang a,b,∗ , Yanlin Zhao b a
Respiratory Diseases Department of Nanlou, Chinese People’s Liberation Army General Hospital, Beijing, People’s Republic of China National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155 Chang Bai Road, Changping District, Beijing 102206, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 13 May 2014 Accepted 13 September 2014 Keywords: Tuberculosis Clofazimine Multidrug resistance Synergy
a b s t r a c t Clofazimine (CLO) is a promising candidate drug for use in the management of multidrug-resistant tuberculosis (MDR-TB) patients. In this study, the minimum inhibitory concentration (MIC) method was used to investigate drug susceptibility to CLO as well as potential synergies between CLO and other antituberculous drugs, including ethambutol (EMB), levofloxacin (LEV), moxifloxacin (MOX), amikacin (AMK) and capreomycin (CAP), among MDR-TB isolates from China. A total of 195 MDR-TB isolates were collected from the national drug resistance survey conducted in China. Of the 195 MDR-TB isolates, 54 (27.7%) were classified as CLO-resistant, whilst 141 (72.3%) were CLO-susceptible with MICs of ≤1 g/mL. In addition, the prevalence of CLO-resistant isolates among the extensively drug-resistant (XDR)-TB group was 61.5% (8/13), which was significantly higher than that of the MDR-TB group (23.0%) (P = 0.006). When fractional inhibitory concentration indexes (FICIs) were calculated for 24 isolates, synergy was found in 11 isolates (45.8%) against the CLO/EMB combination, 6 (25.0%) against the CLO/LEV combination, 8 (33.3%) against the CLO/MOX combination, 4 (16.7%) against the CLO/AMK combination and 5 (20.8%) against the CLO/CAP combination. In addition, 0.5 to ≤2, indifference; and FIC of >2, antagonism [9].
2.4. Data analysis Statistical analysis was performed using SPSS v.14.0 (SPSS Inc., Chicago, IL). Differences were considered to be statistically significant at P < 0.05.
3. Results 3.1. Minimum inhibitory concentrations A total of 195 MDR-TB isolates were included in this study. The MIC distribution of the MDR-TB isolates ranged from 70% of MDR-TB isolates were still susceptible to CLO, suggesting that CLO may serve as a potential candidate for treating MDR-TB in China. In addition, a higher frequency of CLO resistance among XDR-TB isolates was observed in this study. Similar to our findings, Zhang et al. reported that XDR-TB isolates harbour a significantly higher proportion of linezolid resistance compared with MDR-TB isolates [10]. The more active efflux-mediated mechanism and poorer permeability of the mycobacterial cell wall may contribute to the extensive drug resistance, including CLO resistance, among XDRTB strains [10]. The intrinsic resistance mechanisms mentioned above always confer low-level drug resistance, whilst high-level drug resistance is more likely to result from genetic mutations associated with drug resistance [11]. In the present study, 80% of the high-level CLO-resistant isolates (MIC ≥ 16 g/mL) belonged to the XDR-TB group. Hence, we hypothesise that the higher frequency of CLO resistance may also be related to particular CLO-conferring mutations. Unfortunately, the exact mechanism(s) of CLO-mediated antimicrobial activity remains to be established [5]. Whole-genome sequencing of M. tuberculosis isolates with high-level MICs will extend our knowledge on the potential mechanisms conferring CLO resistance in a future study. Multidrug combination chemotherapy containing EMB, FQs and second-line injectable drugs is usually used for the MDR-TB treatment [1]. Thus, we also determined the in vitro activities of these ATDs in combination with CLO against MDR-TB isolates in order to explore the clinical utility of different combination regimens against MDR-TB. Synergies between EMB and other ATDs have been reported for several mycobacterial species [12]. In this study, a synergistic effect is most noted in the combination of EMB and CLO, to which 11 of 24 strains showed synergy. One possible explanation for the synergism may be that EMB increases the permeability of the bacterial envelope by blocking the biosynthesis of cell wall arabinogalactan, thereby enhancing the penetration of other drugs
[11]. In addition, studies have implicated that the mode of action of CLO includes cell-wall-destabilising properties, which may confer an increased influx of EMB [5]. FQs serve as one of the backbone antimicrobial agents for the treatment of MDR-TB. After EMB, MOX showed synergy among one-third of the MDR-TB isolates when combined with CLO. The percentage of synergy among the CLO/LEV combination was lower than that of the CLO/MOX combination, indicating that MOX has better synergistic activity against MDR-TB than LEV when combined with CLO. A second-line injectable drug is always included in the chemotherapy regimen for the treatment of MDR-TB [1]. In the current study of the combined effects of second-line injectable drugs and CLO in vitro, most of the strains showed indifference, whilst synergy was observed for
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short Communication
In vitro synergistic activity of clofazimine and other antituberculous drugs against multidrug-resistant Mycobacterium tuberculosis isolates Zhijian Zhang a,b,1 , Tianzhi Li a,1 , Geping Qu a , Yu Pang a,b,∗ , Yanlin Zhao b a
Respiratory Diseases Department of Nanlou, Chinese People’s Liberation Army General Hospital, Beijing, People’s Republic of China National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155 Chang Bai Road, Changping District, Beijing 102206, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 13 May 2014 Accepted 13 September 2014 Keywords: Tuberculosis Clofazimine Multidrug resistance Synergy
a b s t r a c t Clofazimine (CLO) is a promising candidate drug for use in the management of multidrug-resistant tuberculosis (MDR-TB) patients. In this study, the minimum inhibitory concentration (MIC) method was used to investigate drug susceptibility to CLO as well as potential synergies between CLO and other antituberculous drugs, including ethambutol (EMB), levofloxacin (LEV), moxifloxacin (MOX), amikacin (AMK) and capreomycin (CAP), among MDR-TB isolates from China. A total of 195 MDR-TB isolates were collected from the national drug resistance survey conducted in China. Of the 195 MDR-TB isolates, 54 (27.7%) were classified as CLO-resistant, whilst 141 (72.3%) were CLO-susceptible with MICs of ≤1 g/mL. In addition, the prevalence of CLO-resistant isolates among the extensively drug-resistant (XDR)-TB group was 61.5% (8/13), which was significantly higher than that of the MDR-TB group (23.0%) (P = 0.006). When fractional inhibitory concentration indexes (FICIs) were calculated for 24 isolates, synergy was found in 11 isolates (45.8%) against the CLO/EMB combination, 6 (25.0%) against the CLO/LEV combination, 8 (33.3%) against the CLO/MOX combination, 4 (16.7%) against the CLO/AMK combination and 5 (20.8%) against the CLO/CAP combination. In addition, 0.5 to ≤2, indifference; and FIC of >2, antagonism [9].
2.4. Data analysis Statistical analysis was performed using SPSS v.14.0 (SPSS Inc., Chicago, IL). Differences were considered to be statistically significant at P < 0.05.
3. Results 3.1. Minimum inhibitory concentrations A total of 195 MDR-TB isolates were included in this study. The MIC distribution of the MDR-TB isolates ranged from 70% of MDR-TB isolates were still susceptible to CLO, suggesting that CLO may serve as a potential candidate for treating MDR-TB in China. In addition, a higher frequency of CLO resistance among XDR-TB isolates was observed in this study. Similar to our findings, Zhang et al. reported that XDR-TB isolates harbour a significantly higher proportion of linezolid resistance compared with MDR-TB isolates [10]. The more active efflux-mediated mechanism and poorer permeability of the mycobacterial cell wall may contribute to the extensive drug resistance, including CLO resistance, among XDRTB strains [10]. The intrinsic resistance mechanisms mentioned above always confer low-level drug resistance, whilst high-level drug resistance is more likely to result from genetic mutations associated with drug resistance [11]. In the present study, 80% of the high-level CLO-resistant isolates (MIC ≥ 16 g/mL) belonged to the XDR-TB group. Hence, we hypothesise that the higher frequency of CLO resistance may also be related to particular CLO-conferring mutations. Unfortunately, the exact mechanism(s) of CLO-mediated antimicrobial activity remains to be established [5]. Whole-genome sequencing of M. tuberculosis isolates with high-level MICs will extend our knowledge on the potential mechanisms conferring CLO resistance in a future study. Multidrug combination chemotherapy containing EMB, FQs and second-line injectable drugs is usually used for the MDR-TB treatment [1]. Thus, we also determined the in vitro activities of these ATDs in combination with CLO against MDR-TB isolates in order to explore the clinical utility of different combination regimens against MDR-TB. Synergies between EMB and other ATDs have been reported for several mycobacterial species [12]. In this study, a synergistic effect is most noted in the combination of EMB and CLO, to which 11 of 24 strains showed synergy. One possible explanation for the synergism may be that EMB increases the permeability of the bacterial envelope by blocking the biosynthesis of cell wall arabinogalactan, thereby enhancing the penetration of other drugs
[11]. In addition, studies have implicated that the mode of action of CLO includes cell-wall-destabilising properties, which may confer an increased influx of EMB [5]. FQs serve as one of the backbone antimicrobial agents for the treatment of MDR-TB. After EMB, MOX showed synergy among one-third of the MDR-TB isolates when combined with CLO. The percentage of synergy among the CLO/LEV combination was lower than that of the CLO/MOX combination, indicating that MOX has better synergistic activity against MDR-TB than LEV when combined with CLO. A second-line injectable drug is always included in the chemotherapy regimen for the treatment of MDR-TB [1]. In the current study of the combined effects of second-line injectable drugs and CLO in vitro, most of the strains showed indifference, whilst synergy was observed for
