|Year : 2016 | Volume
| Issue : 4 | Page : 442-447
Drug susceptibility of rapid and slow growing non-tuberculous mycobacteria isolated from symptomatics for pulmonary tuberculosis, Central India
B Goswami1, P Narang1, PS Mishra1, R Narang1, U Narang2, DK Mendiratta1
1 Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, Maharashtra, India
2 Department of Medicine, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, Maharashtra, India
|Date of Submission||01-Aug-2016|
|Date of Acceptance||07-Sep-2016|
|Date of Web Publication||8-Dec-2016|
Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Non-tuberculous mycobacteria (NTM) are emerging as important pathogens. Their treatment also differs from that of Mycobacterium tuberculosis. In India, any datum on them is scarce as species identification and drug susceptibility are not performed in most laboratories. Susceptibility also differs from one geographic area to another, and in our country, there are no data even to guide the clinicians to start treatment empirically.Methodology: The present study endeavours to generate drug susceptibility data on NTM isolated from sputum samples collected and stored from 6445 symptomatics for pulmonary tuberculosis during a prevalence survey and from specimens received from the hospital. Isolates were not necessarily associated with the disease. Species were identified and antibiotic susceptibility was performed using micro-broth dilution technique as per the standard Clinical and Laboratory Standards Institute guidelines. Results: A total of 65 NTM with 11 species were identified, of which 27 belonged to Mycobacterium fortuitum complex, 14 Mycobacterium gordonae, 9 Mycobacterium avium, 7 Mycobacterium flavescens, 4 Mycobacterium scrofulaceum and one each of others. Sensitivity to amikacin for M. fortuitum was 95.22% (20 out of 21), followed by ciprofloxacin (76.19%) and clarithromycin (71.42%). All the 9 M. avium isolates, 11 of M. gordonae (78.57%), 5 of M. flavescens and 2 of M. scrofulaceum were sensitive to clarithromycin. All NTM were resistant to first-line antitubercular drugs except 8, which were sensitive to streptomycin. Conclusions: Drug sensitivity of NTM varies from species to species. While amikacin was the best for rapidly growing mycobacteria, clarithromycin was the most active drug against M. avium and other slow growers.
Keywords: Clinical and Laboratory Standards Institute, drug susceptibility, micro-broth dilution, minimum inhibitory concentration, non-tuberculous mycobacteria
|How to cite this article:|
Goswami B, Narang P, Mishra P S, Narang R, Narang U, Mendiratta D K. Drug susceptibility of rapid and slow growing non-tuberculous mycobacteria isolated from symptomatics for pulmonary tuberculosis, Central India. Indian J Med Microbiol 2016;34:442-7
|How to cite this URL:|
Goswami B, Narang P, Mishra P S, Narang R, Narang U, Mendiratta D K. Drug susceptibility of rapid and slow growing non-tuberculous mycobacteria isolated from symptomatics for pulmonary tuberculosis, Central India. Indian J Med Microbiol [serial online] 2016 [cited 2017 Jan 19];34:442-7. Available from: http://www.ijmm.org/text.asp?2016/34/4/442/195375
| ~ Introduction|| |
Non-tuberculous mycobacteria (NTM) are ubiquitous organisms found in the environment. In India, their importance was first recognised when they were reported to be one of the probable causes for Bacillus Calmette–Guerin vaccine failure in South India. However, today NTM are emerging as important pathogens of both pulmonary and extrapulmonary diseases. Although often unappreciated, worldwide burden of illness with these organisms is substantial, and in industrialised countries, they are more common than Mycobacterium tuberculosis. It is reported that their prevalence is likely to increase globally  as they can cause mycobacteriosis in both immune-compromised as well as immune-competent individuals. Pulmonary manifestations account for almost 94% of these infections  caused predominantly by Mycobacterium avium, Mycobacterium kansasii and Mycobacterium fortuitum complex. Moreover, although the signs and symptoms are similar to tuberculosis, they do not respond to conventional antitubercular treatment  and such cases are very often misdiagnosed clinically as multi-drug-resistant tuberculosis.,, In these cases, acid-fast bacilli on the smear cannot be taken as the only representative of M. tuberculosis. Culture is necessary to identify and speciate the isolate because therapy has to be optimised according to the species.
Looking into the fact that NTM are also colonisers of the respiratory tract, the American Thoracic Society  has advocated repeat cultures of the sputum to establish pulmonary disease; however, this norm is not followed by most of the laboratories in India, and repeat sample is seldom requisitioned. The NTM isolate from a single sputum sample is considered as contaminant, and as a result, the exact magnitude of the problem never gets assessed. NTM are definitely more prevalent than believed because, in the year 2000, the National AIDS Control Organization in one of its Specialist Training and Reference Module  had mentioned that M. avium complex (MAC) which is prevalent in the west does not exist in India; however, subsequently, two studies reported for the first time that not only does it exist  but it also causes disseminated disease in HIV-seropositive patients. Some more reviews,, prospective , and retrospective , studies confirmed their importance. But literature on NTM and particularly on their susceptibility to drugs in India continues to be extremely scarce. It is well recognised that there is geographic variation not only in the prevalence of the species but also in their drug susceptibility. Ideally, every clinical isolate in the laboratory should be subjected to drug susceptibility test (DST), but in India, when most laboratories are still struggling to establish DST for M. tuberculosis, the same for NTM is too much to expect. It is, therefore, imperative that laboratories working on NTM should generate data and disseminate information that can help the clinicians to manage their patients even in the absence of immediate sensitivity report.
Accordingly, the present study was taken up to perform and report on drug susceptibility of some prevalent NTM, irrespective of their association with disease, though the NTM were isolated from the sputum of subjects with pulmonary symptoms.
| ~ Methodology|| |
The study, after getting the approval from the Institutional Ethics Committee, was conducted from 2012 to 2013 in the mycobacteriology laboratory of a medical college situated in the rural area of a district in Central India. The laboratory is accredited under the Revised National Tuberculosis Control Programme for first-line antituberculosis drugs on solid media. The study consisted of reviving and processing the non-duplicate NTM isolates stocked in glycerol 7H9 broth at −70°C during the TB prevalence survey conducted by the department in the district from 2007 to 2009. In the survey, 12,890 sputum samples from 6445 symptomatics for pulmonary tuberculosis were processed. It also included hospital isolates recovered during the study period. All NTM were speciated based on the conventional growth and biochemical characters.
DST was done as per the Clinical and Laboratory Standards Institute (CLSI) guidelines  using micro-broth dilution method. Muller-Hinton broth was used for rapidly growing mycobacteria (RGM) and Middlebrook 7H9 broth for slowly growing mycobacteria (SGM). The drug concentrations and minimum inhibitory concentration (MIC) break point for RGM and MAC were as per CLSI, whereas for SGM, other than MAC, Heifets  break points were used. Owing to the paucity of funds, not all drugs recommended by CLSI were used, but in addition, first-line antitubercular drugs, though not recommend by CLSI, were included for all the isolates.
Control strain for RGM was Staphylococcus aureus ATCC 29213, whereas for M avium, the standard control strain (JALMA T-4) procured from the National Mycobacterial Repository at National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, was used. Currently, CLSI does not recommend, other than for rifampicin, any optimal reference strain for slow growing NTM; therefore, in-house strains confirmed by Line probe assay/high-performance liquid chromatography were included as controls after repeated DST confirmed their MIC and reproducibility.
The drugs procured from Sigma Chemicals were dissolved in distilled water for preparation of the stock solution, except for rifampicin where dimethylformamide was used as diluent. For all drugs, concentrations ranging from 0.125 to 128 µg/ml were prepared, and using one horizontal row for each drug, the concentrations were, respectively, dispensed as 100 µl in each well of the 96-well U-bottomed microtitre plate (Tarsons, India). Then, 100 µl of the strain (turbidity 0.5 McFarland's standard) was added to each well in the row including the last empty control well. One separate row for each drug was used for the control strain. Plates were then sealed with paraffin film and put in plastic bags and incubated at 37°C.
Plates were read using inverted mirror. A bead up to 2 mm in the control well indicated good growth and MIC was defined as the lowest concentration of the drug that inhibited visible growth. In case of sulfamethoxazole, the end-point was taken as 80% inhibition of growth compared to control well. For RGM, reading was taken after 72 h whereas SGM were read at 7 days and finally at 14 days of incubation.
| ~ Results|| |
In total, 65 isolates within 11 species of NTM were recovered, of which 34 were from stock of TB prevalence survey and 31 were from symptomatic patients reporting to the hospital. Of these 65, 21 (32.30%) isolates were of M. fortuitum, 3 (4.61%) M. chelonae, 3 (4.61%) M. abscessus, 14 (21.53%) M. gordonae, 9 (13.84%) M. avium, 7 (10.76%) M. flavescens and 4 (6.1%) M. scrofulaceum. Rest of the isolates were one each of M. kansasii, M. simiae, M. gastri and M. triviale.
[Table 1] shows that all the isolates of M. fortuitum complex were resistant to all first-line antitubercular drugs except streptomycin, whereas 3 isolates of M. fortuitum and 1 isolate of M. abscessus were sensitive. Of the 21 isolates of M. fortuitum tested, 20 (95.22%) were sensitive to amikacin within the MIC range of 4–16 µg/ml; 16 (76.19%) to ciprofloxacin, MIC range of 0.5–1 µg/ml and 15 (71.42%) to clarithromycin, MIC range of 1–2 µg/ml. Of the three strains of M. chelonae tested, all were susceptible to amikacin within MIC range of 8–16 µg/ml. Two strains of M. abscessus were sensitive to amikacin, clarithromycin and ciprofloxacin with MIC of 16 µg/ml, 1–2 µg/ml and 1 µg/ml, respectively. The overall susceptibility of these three species of fortuitum complex was 88.88% to amikacin, 70.37% to ciprofloxacin and 66.66% to clarithromycin. Sensitivity to sulfamethoxazole and doxycycline was poor, 29.62% and 21.57%, respectively.
|Table 1: Drug susceptibility of 27 Mycobacterium fortuitum complex with minimal inhibitory concentration levels in βg/ml|
Click here to view
All isolates of M. avium [Table 2] were resistant to first-line ATT drugs except 2 strains which were in the intermediate range of sensitivity to ethambutol. They were all sensitive to clarithromycin in the range of 2–8 µg/ml.
|Table 2: Drug susceptibility result of nine Mycobacterium avium with minimal inhibitory concentration levels in βg/ml|
Click here to view
Of the 14 strains of M. gordonae tested [Table 3], 11 (78.57%) were sensitive to clarithromycin in the MIC range of 2–8 µg/ml; 10 (71.42%) to amikacin, MIC range of 4–16 µg/ml; 6 (42.85%) to sulfamethoxazole in the MIC range of 16–32 µg/ml; 4 (28.57%) to ciprofloxacin with MIC at 2 µg/ml and 3 (21.42%) to doxycycline with MIC at 4 µg/ml. All isolates were resistant to first-line antitubercular drugs except streptomycin, to which 4 strains were sensitive in the MIC range of 1–2 µg/ml.
|Table 3: Drug Susceptibility result of 14 Mycobacterium gordonae isolates minimal inhibitory concentration levels in βg/ml|
Click here to view
Of the 7 strains of M. flavescens tested [Table 4], 5 (71.4%) were sensitive to clarithromycin in the MIC range of 1–8 µg/ml; 4 (57.1%) to amikacin, MIC range of 4–16 µg/ml; 3 (42.8%) to sulfamethoxazole in the MIC range of 4–16 µg/ml and only 1 (14.2%) to ciprofloxacin at 1 µg/ml. M. scrofulaceum isolates [Table 4] were relatively more resistant with only 2 out of 4 being sensitive to clarithromycin and amikacin and one to ciprofloxacin.
|Table 4: Drug sensitivity of Mycobacterium flavescens and Mycobacterium scrofulaceum with minimal inhibitory concentration in β/ml|
Click here to view
| ~ Discussion|| |
There are more than 170 species of NTM reported in literature, and globally, there is considerable variation in their geographical distribution. In industrialised countries, these are organisms of importance, whereas in developing countries, the work and research on these organisms is on extremely low priority, probably because of the existing burden of M. tuberculosis and limited availability of rapid molecular methods. In spite of these, one report  has quoted several examples from Nigeria, Zambia, Burkina Faso, Mali and Iran where NTM have shown a rise in incidence, and the infections have been misdiagnosed and mistreated with antitubercular drugs, to which these organisms are resistant.
In India, there are only a handful of studies ,,,,,,, that have actually addressed the problem of NTM and there is hardly any datum on their susceptibility to drugs.,, CLSI advocating the use of micro-broth dilution method for testing also came out with the guidelines only in 2003 (M-24A).
CLSI does not recommend DST for first-line antitubercular drugs. However, in the present study, these drugs were included because one of the studies from India  had reported a fair amount of sensitivity of MAC isolates to rifampicin, isoniazid, ethambutol and streptomycin. All our NTM showed complete resistance to all drugs except to streptomycin, to which negligible susceptibility was noted.
Susceptibility profiles of NTM vary greatly according to species and geographical areas. Our isolates of M. fortuitum and M. chelonae from Central India showed 100% sensitivity to amikacin, but only two out of the three strains of M. abscessus tested were sensitive. A study from South India, while testing 66 isolates of M. fortuitum, has also reported 100% sensitivity to the drug. One hundred percent sensitivity to amikacin was reported for all isolates from Taiwan  when 69 M. fortuitum, 39 M. chelonae and 92 M. abscessus were tested. The authors recommended that in the absence of laboratory report, amikacin can be selected as the drug of choice for RGM.
Swenson et al. from CDC, Atlanta, have also reported 100% sensitivity of M. fortuitum to amikacin and have stated that this drug attains good blood serum levels. On the other hand, taking lower break point of 5 mg/L and using agar dilution method, The Netherlands' study  has reported 56% sensitivity for M. fortuitum but M. chelonae and M. abscessus were reported as highly resistant. Swenson et al. have advocated two oral drugs, doxycycline and sulfamethoxazole, as other agents for treatment, but our strains showed poor sensitivity to both these drugs.
For M. fortuitum complex, we found clarithromycin to be the next best drug, followed by ciprofloxacin; however, in Taiwan, the sensitivity of M. abscessus to these drugs was extremely poor. The sensitivity of The Netherlands' isolates to clarithromycin was better than amikacin.
The above reports highlight the fact that there is a substantial variation in sensitivity profile within the species and from one geographical area to another.
The introduction of macrolides, especially clarithromycin, has greatly enhanced the treatment of NTM  and is also recommended by CLSI as the class drug for M. avium. All our M. avium isolates were sensitive to the drug. There are no reports available in our country on the clinical use and response to clarithromycin, but resistance to macrolides is now well documented, and if this resistance is to be countered, multidrug regimens particularly with rifabutin and ethambutol  need to be evaluated clinically.
Among the other slow growers, we tested 14 M. gordonae, 7 M. flavescens and 4 M. scrofulaceum. All the isolates showed the best sensitivity to clarithromycin, followed by amikacin. Brown et al. from Texas have also reported all their 6 strains of M gordonae and 5 M. scrofulaceum as fully sensitive to clarithromycin. High sensitivity to this drug was also reported from The Netherlands, but the same study has reported all their 8 isolates of M. scrofulaceum to be resistant to amikacin. Globally, the data on SGM are scarce, and M. gordonae is generally considered as the least pathogenic among the environmental NTM although some cases of infections , have been reported. There is no Indian literature on the drug susceptibility of these three SGM, and our report, though on small numbers, is probably the first in the country.
| ~ Conclusions|| |
India is rapidly moving towards control of tuberculosis. NTM, being resistant to antitubercular drugs, are sure to emerge as important pathogens, particularly when India continues to harbour heavy burden of patients with HIV/AIDS. Industrialised countries have witnessed this phenomenon. It is hoped that the data generated from this small study will not only help the clinicians but also motivate more laboratories in India to work on NTM and prepare for the future.
We acknowledge the financial assistance for the study provided by the Kasturba Health Society, Sevagram, and the Revised National Tuberculosis Control Program, Wardha District. We also acknowledge the National Repository for Mycobacterial isolates at the National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra, for kindly providing the control strains for the study.
Financial support and sponsorship
This study was supported by the Kasturba Health Society, Sevagram, the Revised National Tuberculosis Control Program, Wardha District.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Paramasivan CN, Govindan D, Prabhakar R, Somasundaram PR, Subbammal S, Tripathy SP. Species level identification of non-tuberculous mycobacteria from South Indian BCG trial area during 1981. Tubercle 1985;66:9-15.
Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al
. An official ATS/IDSA statement: Diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175:367-416.
Raju RM, Raju SM, Zhao Y, Rubin EJ. Leveraging advances in tuberculosis diagnosis and treatment to address nontuberculous mycobacterial disease. Emerg Infect Dis 2016;22:365-9.
O'Brien RJ, Geiter LJ, Snider DE Jr. The epidemiology of nontuberculous mycobacterial diseases in the United States. Results from a national survey 1. Am Rev Respir Dis 1987;135:1007-14.
Pokam BT, Asuquo AE. Acid-fast bacilli other than mycobacteria in tuberculosis patients receiving directly observed therapy short course in Cross River State, Nigeria. Tuberc Res Treat 2012;2012:301056.
Maiga M, Siddiqui S, Diallo S, Diarra B, Traoré B, Shea YR, et al
. Failure to recognize nontuberculous mycobacteria leads to misdiagnosis of chronic pulmonary tuberculosis. PLoS One 2012;7:e36902.
Shahraki AH, Heidarieh P, Bostanabad SZ, Khosravi AD, Hashemzadeh M, Khandan S, et al
. “Multidrug-resistant tuberculosis” may be nontuberculous mycobacteria. Eur J Intern Med 2015;26:279-84.
National AIDS Control Organization. Specialists' training and reference module. New Delhi: Ministry of Health and Family Welfare; 2000. p. 108.
Narang P, Narang R, Bhattacharya S, Mendiratta DK. Paraffin slide culture technique for isolating non-tuberculous mycobacteria from stool and sputum of HIV sero-positive patients. Indian J Tuberc 2004;51:23-6.
Narang P, Narang R, Mendiratta DK, Roy D, Deotale V, Yakrus MA, et al
. Isolation of Mycobacterium avium
complex and M. simiae
from blood of AIDS patients from Sevagram, Maharashtra. Indian J Tuberc 2005;52:21-6.
Katoch VM. Infections due to non-tuberculous mycobacteria (NTM). Indian J Med Res 2004;120:290-304.
Gopinath K, Singh S. Non-tuberculous mycobacteria in TB-endemic countries: Are we neglecting the danger? PLoS Negl Trop Dis 2010;4:e615.
Singh S, Gopinath K, Shahdad S, Kaur M, Singh B, Sharma P. Nontuberculous mycobacterial infections in Indian AIDS patients detected by a novel set of ESAT-6 polymerase chain reaction primers. Jpn J Infect Dis 2007;60:14-8.
Narang R, Narang P, Jain AP, Mendiratta DK, Joshi R, Lavania M, et al
. Disseminated disease caused by Mycobacterium simiae
in AIDS patients: A report of three cases. Clin Microbiol Infect 2010;16:912-4.
Jesudason MV, Gladstone P. Non tuberculous mycobacteria isolated from clinical specimens at a tertiary care hospital in South India. Indian J Med Microbiol 2005;23:172-5.
Myneedu VP, Verma AK, Bhalla M, Arora V, Reza S, Sah GC, et al
. Occurrence of Non-tuberculous Mycobacterium
in clinical samples – A potential pathogen. Indian J Tuberc 2013;60:71-6.
Winn WC, Allen SD, Allen SA, Janda WM, Koneman EW, Schreckenberger PC. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 6th
edition. Lippincott Williams & Wilkins Wolters Kluwer Company, Baltimore & Philadelphia, USA 2006.
Clinical and Laboratory Standards Institute. Susceptibility Testing of Mycobacteria, Nocardiae and Other Aerobic Actinomycetes; Approved Standard CLSI Documents M24-A2. 2nd
ed. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute; 2011.
Heifets L. Susceptibility testing of Mycobacterium avium
complex isolates. Antimicrob Agents Chemother 1996;40:1759-67.
Gayathri R, Therese KL, Deepa P, Mangai S, Madhavan HN. Antibiotic susceptibility pattern of rapidly growing mycobacteria. J Postgrad Med 2010;56:76-8.
Venugopal D, Kumar S, Isa M, Bose M. Drug resistance profile of human Mycobacterium avium
complex strains from India. Indian J Med Microbiol 2007;25:115-20.
Sankar MM, Gopinath K, Singla R, Singh S. In-vitro
antimycobacterial drug susceptibility testing of non-tubercular mycobacteria by tetrazolium microplate assay. Ann Clin Microbiol Antimicrob 2008;7:15.
Yang SC, Hsueh PR, Lai HC, Teng LJ, Huang LM, Chen JM, et al
. High prevalence of antimicrobial resistance in rapidly growing mycobacteria in Taiwan. Antimicrob Agents Chemother 2003;47:1958-62.
Swenson JM, Thornsberry C, Silcox VA. Rapidly growing mycobacteria: Testing of susceptibility to 34 antimicrobial agents by broth microdilution. Antimicrob Agents Chemother 1982;22:186-92.
van Ingen J, van der Laan T, Dekhuijzen R, Boeree M, van Soolingen D.In vitro
drug susceptibility of 2275 clinical non-tuberculous Mycobacterium
isolates of 49 species in the Netherlands. Int J Antimicrob Agents 2010;35:169-73.
Nash KA, Inderlied CB. Genetic basis of macrolide resistance in Mycobacterium avium
isolated from patients with disseminated disease. Antimicrob Agents Chemother 1995;39:2625-30.
Brown BA, Wallace RJ Jr., Onyi GO. Activities of clarithromycin against eight slowly growing species of nontuberculous mycobacteria, determined by using a broth microdilution MIC system. Antimicrob Agents Chemother 1992;36:1987-90.
Foti C, Sforza V, Rizzo C, De Pascale G, Bonamonte D, Conserva A, et al
. Cutaneous manifestations of Mycobacterium gordonae
infection described for the first time in Italy: A case report. Cases J 2009;2:6828.
Mazumder SA, Hicks A, Norwood J. Mycobacterium gordonae
pulmonary infection in an immunocompetent adult. N Am J Med Sci 2010;2:205-7.
[Table 1], [Table 2], [Table 3], [Table 4]