|Year : 2002 | Volume
| Issue : 3 | Page : 137-140
A preliminary report on characterization and identification of non tuberculous mycobacteria (NTM) on the basis of biochemical tests and protein / isoenzyme electrophoretic patterns
P Gupta , VM Katoch , UD Gupta , DS Chauhan , R Das , D Singh , K Srivastava , HB Singh , VD Sharma
Central JALMA Institute for Leprosy (ICMR), Tajganj, Agra - 282 001, India
Central JALMA Institute for Leprosy (ICMR), Tajganj, Agra - 282 001, India
PURPOSE: To assess the usefulness of protein electrophorograms and protein zymodemes in the identification and characterization of non tuberculous mycobacteria (NTM). METHODS: Cell free extracts (CFEs) from 22 mycobacterial isolates belonging to slow growing and other clinically relevant species were included in the study. The strains isolated from the environment were identified on the basis of their standard biochemical tests; pigmentation and growth characters. The CFEs were electrophoresed and stained for proteins and esterases. RESULTS: Most of the isolates identified on the basis of biochemical tests exhibited characteristic protein and esterase pattern for M.scrofulaceum, M.avium and M.xenopi. Others showed variations in their proteins and esterase pattern though they were identified as M.scrofulaceum, M.avium and M.xenopi. CONCLUSIONS: Based on these studies it appears that because of variability in the protein and isoenzyme patterns of NTM, it may be advisable to use them along with biochemical tests and other tests for identifying and characterizing the different mycobacterial species belonging to slow growers.
|How to cite this article:|
Gupta P, Katoch V M, Gupta U D, Chauhan D S, Das R, Singh D, Srivastava K, Singh H B, Sharma V D. A preliminary report on characterization and identification of non tuberculous mycobacteria (NTM) on the basis of biochemical tests and protein / isoenzyme electrophoretic patterns. Indian J Med Microbiol 2002;20:137-40
|How to cite this URL:|
Gupta P, Katoch V M, Gupta U D, Chauhan D S, Das R, Singh D, Srivastava K, Singh H B, Sharma V D. A preliminary report on characterization and identification of non tuberculous mycobacteria (NTM) on the basis of biochemical tests and protein / isoenzyme electrophoretic patterns. Indian J Med Microbiol [serial online] 2002 [cited 2020 Sep 30];20:137-40. Available from: http://www.ijmm.org/text.asp?2002/20/3/137/6937
Mycobacteria other than M.tuberculous complex (MOTT) mainly exist in the environment as saprophytes and have been recognized as a cause of human disease for a very long time. In the past these were called atypical mycobacteria but later the name non - tuberculous mycobacteria (NTM) has been given to them. Diseases caused by these organisms are uncommon compared with tuberculosis but there has been a significant increase in the pulmonary and non-pulmonary infections due to these mycobacteria during the last two to three decades.,,,
There has been slow and steady increasing interest in the use of protein and isoenzyme patterns in identification of various non tuberculous mycobacteria during last 50 years.,,,
The isolation and identification of mycobacteria from the clinical specimens often requires several weeks before the results are available. The present methods of identification of mycobacterial species are based on various biochemical reactions which are time consuming and the variations cause difficulty in the interpretations of the results. Protein electrophorograms / isoenzyme patterns, lipid patterns and gene probes are alternate methods for identification of mycobacteria. Limited studies about the application of the proteins and isoenzyme pattern analysis for characterization of mycobacteria are available in the literature.,,,,,, Sharma et al, described species and strain specific pattern of various slow and rapid growing mycobacteria and based on their ease / rapidity of application recommends them for clinical application. The scheme proposed in these studies needs to be investigated for its usefulness in characterizing isolates in clinical mycobacterial laboratories. As environment is the usual source of infection due to NTM, in the present study an effort has been made to characterize and identify some mycobacterial isolates based on biochemical tests and correlate with the analysis of protein and esterase patterns.
| ~ Materials and Methods|| |
In this study, 22 mycobacterial isolates belonging to slow growing and clinically relevant species (isolated from the environment) were included. Various biochemical tests like nitrate reduction, niacin production, catalase, arylsulphatase, tween 80-hydrolysis, pyrazinamidase, urease etc. and pigmentation were done as per standard procedures. Growth of mycobacteria and preparation of cell free extract was done as per the procedure described by Katoch et al. Cell free extracts were subjected to discontinuous 7.5% PAGE as described earlier.,, The gels were visually examined for enzymes and proteins using Coomassie Brilliant Blue G - 250 and for esterases by the method of Lawrence et al. The gels were visually examined for enzymes and prominent protein bands and their electrophoretic mobilities (eF) were calculated as described earlier.,, The zymograms and protein electrophorograms based on eF values of the bands of the individual strain were constructed and grouped. Differences in eF values of 0.05 in same / opposite direction for bands was considered as insignificant.
| ~ Results|| |
The results of biochemical tests are presented in [Table - 1]. On the basis of biochemical tests, these 22 isolates can be classified in to three species - M.scrofulaceum, M.avium and M.xenopi .
Out of 22 isolates, 14 isolates showed all the characteristics of M.scrofulaceum but one strain showed some variation with respect to tween-80 hydrolysis. Similarly in only one isolate, all characteristics of M.avium were observed but in 4 isolates variations were observed with respect to tween-80 hydrolysis, pyrazinamidase and arylsulfatase tests. Three isolates showed characteristics similar to M.xenopi but there was a lot of variability in strains with respect to tween-80 hydrolysis, urease and pyrazinamidase.
| ~ Discussion|| |
Our observations in this study indicate that Tween-80 and pyrazinamidase, though important tests, have technical limitations due to variability. Such variability may lead to misidentification of mycobacteria at times.
The eF values of proteins of different isolates are presented in [Figure - 1]. From the figure, it is clear that based on the protein patterns ICC 178 (eF values - 0.2, 0.32,0.33,0.38,0.45,0.53,0.64,0.67,0.70,0.85 & 0.92), ICC 180 (eF values - 0.15,0.18,0.30,0.70,0.80,0.92), ICC 188 (eF values - 0.15,0.30,0.40,0.50,0.70,0.80 & 0.92) and ICC 198 (eF values - 0.30,0.40,0.50,0.78,0.82 & 0.92) appear to resemble the species specific pattern of M. scrofulaceum as bands of J-21 with eF values 0.15,0.20,0.40,0.70,0.80 & 0.92 are mostly present in the above mentioned isolates (underlined) while other isolates, though appear to be M.scrofulaceum biochemically exhibited different patterns. It needs to be ascertained if these belong to different unknown species or subspecies.
Similarly ICC 177 and ICC 183 appear to resemble the species specific patterns of M.avium but ICC 192 and ICC 194 though showing similarities to M.avium show different patterns. As MAIS - Mycobacterium avium intracellulare scrofulaceum complex is not a compact cluster, these strains may belong to different subgroups within this cluster comprising of M.scrofulaceum, M.intracellulare, M.avium and many intermediate groups. This can be ascertained by rRNA sequencing or probe hybridization. Of the three isolates identified as M.xenopi based on biochemical tests, ICC 190 showed resemblance to species specific pattern of M.avium but ICC 186 and ICC 202 may belong to unknown new subgroups.
The esterase patterns of different isolates are presented in [Figure:2]. The isolates ICC 181 and ICC 187 exhibited 3 bands and their eF appear to resemble to the previously reported patterns of Sharma et al as well as the Control J-7 and N-25 used in this study. There appears to be two new subgroups within isolates identified as M.scrofulaceum (based on biochemical tests) - (a) One group with single band with eF value around 0.93-0.98 (ICC-185, ICC-189, ICC-191, ICC-195) (b) Other group with 2 bands with eF value ranging between 0.82 to 0.98 (ICC 178, ICC 179 and ICC 182). On the other hand ICC 177 and ICC 192 identified as M.avium showed 1 band (eF value = 0.83) and 2 bands (eF values = 0.80 & 0.95) respectively which are in conformity with the report of Sharma et al who have also reported 1 and 2 bands respectively in strains of M.avium. Further, there appears to be one more subgroup exhibiting 3 bands (ICC 199 & ICC 194) which appear like M.avium, but may belong to some unknown new subgroups. In the isolates identified as M.xenopi, ICC 190 showed 2 bands like Sharma et al with eF values of 0.76 & 0.92 respectively. However, ICC 186 and ICC 202 exhibited 5 bands with eF values ranging between 0.57 - 0.91.
From the above studies, it is clear that though most of the isolates are showing biochemical tests and protein and esterases patterns like M.scrofulaceum, M.avium and M.xenopi, certain strains showed variations in biochemical characters but showed similar protein patterns (e.g. ICC 177 and ICC 183). On the other hand strains like ICC 186 and ICC 202 showed variations in biochemical tests as well as in proteins and esterase patterns and are not in conformity with the earlier reports. This may be due to very small number of isolates / strains taken in the earlier studies, single isoenzyme investigated as well as due to differences in geographical sources of the isolates (Indian isolates in present study compared to strains from Western world in earlier studies).
It is concluded that even though there is lot of variability in the protein and isoenzyme patterns of NTM, they alone or along with biochemical tests can be useful in characterizing and identifying the different mycobacterial species / subspecies / strains. However, these results need to be correlated with modern molecular techniques like species / strain specific probes and rRNA sequencing. Strict conditions for their applications will have to be kept in mind as expression may vary. During the recent years several new species of environmental mycobacteria have also been identified using newer taxonomical techniques, and therefore, in depth study is required to characterize these environmental as well as clinical isolates. Comparative ease of applications of these methods makes them an attractive alternative candidates in situations with limited resources.
| ~ Acknowledgement|| |
Financial support from Department of Biotechnology (DBT) to Mycobacterial Repository Centre (Grant No. BT / 03 / 04 / 94 PID) is gratefully acknowledged.
| ~ References|| |
|1.||Duvall CW. Studies on a typical forms of tubercle bacilli isolated directly from human cases of primary cervical adenitis. J Exp Med 1908;9:403-429. |
|2.||Pinner A. Atypical acid fast microrganisms. Am Rev Tubercl 1935;32: 424-445. |
|3.||Wolinsky E, Rynearson TK. Mycobacteria in soil and their relation to disease producing strains. Am Rev Resp Dis 1968;97:1032-1037. |
|4.||Wolinksy E. Non tuberculous mycobacteria and associated diseases. Am Rev Resp Dis 1979;119:107-159. |
|5.||Good RC. Opportunistic pathogens in the genus Mycobacterium. Ann Rev Microbiol 1985;39:347-369. |
|6.||Wayne LG, Sramek HA. Agents of newly recognized or infrequently encountered mycobacterial diseases. Clin Microbiol Rev 1992;5:1-25. |
|7.||Nakayama Y. The electrophoretic analysis of esterases and catalase and its use taxonomy of mycobacteria. Jap J Microbiol 1967;11:95-101. |
|8.||Haas H, Davidson Y, Sacks T. Taxonomy of mycobacteria by polyacrylamide gel electrophoresis of cell protein. J Med Microbiol 1972;5:31-37. |
|9.||Vrba M, Zampa P. Interspecies variation of tetrazolium oxidase electrophoresis of cell protein. Folio-Microbol 1973;18:32-39. |
|10.||Katoch VM, Sharma VD, Datta AK, Shivannavar C T, Kannan KB, and Bharadwaj VP. Isozyme patterns of mycobacteria in taxonomical identification of mycobacteria, specially M.leprae. Indian J Med Microbiol 1986;4:81-87. |
|11.||Dhandayuthapani S, Nalini N, Bhatia VN. LDH isozyme as a tool for identification of mycobacteria. Indian J Lepr 1989;61:61-64. |
|12.||Sharma VD, Katoch VM, Shivannavar C T, Gupta UD, Sharma RK, Patil MA, Bharadwaj V P, Agarwal B M . Protein and isozyme patterns of mycobacteria - 1. Their role in rapid identification of rapidly growing mycobacteria. Indian J Med Microbiol 1995;13:115-118. |
|13.||Sharma VD, Katoch V M, Shivannavar C T, Gupta U D, Sharma RK, Patil M A, Bharadwaj V P, Agarwal BM. Protein and isozyme patterns of mycobacteria - II. Their role in rapid identification of slow growing mycobacteria. Indian J Med Microbiol 1995;13:119-123. |
|14.||Vestal AL. Procedure for the isolation and identification of mycobacteria. Centre for Disease Control, Atlanta, USA, 1977. |
|15.||Davis BJ. Disc Electrophoresis. II. Method and application to human serum protein. Ann New York Acad Sci 1964;121:404-427. |
|16.||Smith I. In "Chromatographic and Electrophoretic techniques". (William Heineman Medical Books, London). 1976,Vol II:153 & 321. |
|17.||Holbrook IB, Leaver AG. A procedure to increase the sensitivity of staining by Coomassie Brilliant Blue G-250 perchloric acid solution. Anal Biochem 1976;75:634-636. |
|18.||Lawrence SH, Melnick PJ, Wekner HE. Proc. Soc. Exp. Biol. Med. 1960; 105:572. In : Methods in Microbiology. Vol. 5B. Norris JR and Ribbon D W, Eds (Academic Press, London New York) 1971:590. |
|19.||Butler WRL, Thibert MI, Kilburn JO. Identification of Mycobacterium avium complex strains and some similar species by high performance liquid chromatography. J Clin Microbiol 1992;30:2698-2704. |
|20.||Bottger EC, Hirschel B, Coyle MB. Mycobacterium genavense sp. Nov. Int J Clin Microbiol 1993;43:841-843. |