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 ~  Abstract
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  Acknowledgement
 ~  References

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Year : 2004  |  Volume : 22  |  Issue : 4  |  Page : 238-240
 

DNA fingerprinting of Mycobacterium Tuberculosis isolates from Agra region by is 6110 probe


Department of Microbiology and Molecular Biology, Central JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra 282 001, Uttar Pradesh, India

Date of Submission19-Sep-2003
Date of Acceptance22-Dec-2003

Correspondence Address:
Department of Microbiology and Molecular Biology, Central JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra 282 001, Uttar Pradesh, India

 ~ Abstract 

DNA fingerprinting using IS 6110 probe has been used all over the world quite successfully to characterize M. tuberculosis strains. The present study has been carried out to study the polymorphism among isolates of M.tuberculosis from Agra region from patients attending the clinics at SN Medical College and TBDTC, Agra. Sputa were collected in sterilized containers and brought to CJIL, Agra. Samples were processed and cultured on Lowenstein Jensen (LJ) slants. M. tuberculosis isolates were identified by standard biochemical tests. DNA from these isolates were purified by a physicochemical procedure, restricted with Pvu II enzyme and hybridized with PCR amplified and DIG labeled 245 bp IS 6110 probe. With a view to study IS 6110 polymorphism, M. tuberculosis isolates obtained from different geographical areas of Agra region were analyzed. Among the 60 isolates taken in study, 5 had no copy of IS 6110, 8 had 1-4 copies and 47 had multiple copies of IS 6110. DNA fingerprinting using this probe was found to be quite discriminating for typing of most of the strains (80%) which had multiple copies. RFLP profiles did not correlate with geographical areas, contacts or the resistance pattern of the strains. While this data shows the potential of IS 6110 based RFLP for strain characterization of M.tuberculosis in Agra, to understand the molecular epidemiology of tuberculosis in this region, a larger number of isolates from defined geographical areas need to be studied.

How to cite this article:
Chauhan A, Chauhan D S, Parashar D, Gupta P, Sharma V D, Sachan A S, Gupta R, Agarawal B M, Katoch V M. DNA fingerprinting of Mycobacterium tuberculosis isolates from Agra region by is 6110 probe. Indian J Med Microbiol 2004;22:238-40


How to cite this URL:
Chauhan A, Chauhan D S, Parashar D, Gupta P, Sharma V D, Sachan A S, Gupta R, Agarawal B M, Katoch V M. DNA fingerprinting of Mycobacterium tuberculosis isolates from Agra region by is 6110 probe. Indian J Med Microbiol [serial online] 2004 [cited 2019 Oct 18];22:238-40. Available from: http://www.ijmm.org/text.asp?2004/22/4/238/12814


According to a WHO report, India harbours more than 30% of world's cases of human tuberculosis.[1] Keeping in view the global prevalence of tuberculosis and pathogenic importance of M. tuberculosis, there is an urgent need to evolve and apply techniques that not only rapidly identify but also characterize tubercle bacilli to facilitate epidemiological studies. Investigations on the epidemiology of tuberculosis need strain specific markers, which can be used to differentiate M. tuberculosis isolates. DNA based technology is now available for molecular characterization of M. tuberculosis and other mycobacteria. The discovery of a variety of repetitive DNA elements in M. tuberculosis genome[2] has led to the development of restriction fragment length polymorphism (RFLP) methods for differentiation of clinical isolates of M. tuberculosis.
The most widely utilized method of DNA fingerprinting uses the insertion sequence IS 6110 to visualize DNA RFLP of M. tuberculosis.[3] IS 6110 fingerprinting has proven useful for investigating nosocomial transmission, investigating outbreaks, confirming instances of laboratory cross contamination, differentiating relapse caused by endogenous reactivation from re-infection by an exogenous strain and studying TB transmission in large populations.[4],[5],[6] There are a limited number of studies on DNA fingerprinting of Indian M. tuberculosis strains.[7],[8],[9],[10],[11],[12] It would be important to gain experience about application of various molecular typing method to isolates from different parts of country. In the present study, we have investigated IS 6110 polymorphism in M. tuberculosis isolates from Agra region and attempted to correlate the RFLP patterns based on IS 6110 copy numbers with drug susceptibility or resistance profile.

 ~ Materials and Methods Top

Pulmonary tuberculosis patients with sputa positive for acid fast bacilli (AFB) attending outpatient department (OPD) of SN Medical College, and TB Demonstration and Training Centre (TBDTC), Agra, were selected for the study. A total of 226 cases were studied during a period of 2 years (Feb. 2001-March 2003). These included patients with case history of treatment failure/relapse, treatment less than 3 months and fresh cases. Sputum samples were collected in sterilized containers and were processed by modified Petroff's method using 4% NaOH and cultured on Lowenstein Jensen (LJ) slants. On observation of growth in 115 slants, a series of biochemical tests including niacin, nitrate reduction, catalase at 68C, tween 80 hydrolysis and aryl sulphatase were performed for the confirmation of M.tuberculosis.[13] Of the 115 isolates, 80 were confirmed as M.tuberculosis. History of previous treatment and a low number of viable bacilli in sputum of patients undergoing treatment could be a possible reason for the poor isolation rate of mycobacteria. Cells were scraped from LJ slants and DNA was isolated.[3]
A 245 bp probe was amplified by PCR using primers INS 1 (5' C G T G A G G G C A T C G A G G T G G C 3') and INS 2 (5' G C G T A G G C G T C G G T G A C A A A 3') as described by van Soolingen et al.[3],[4] Probe was labelled with non radioactive digoxigenin by the random primed DNA labeling technique using DIG DNA labelling and detection kit (Roche Diagnostics, Germany). One to two microgram of intact DNA was digested with 20 units of restriction enzyme Pvu II (Bangalore genei, India) for 4 hours at 37C in a shaking water bath, and electrophoresed on a 1% agarose gel for 8.5 hours at 1.2 V/cm. The DNA samples were southern blotted on to positively charged nylon membrane (Roche Diagnostics, Germany) overnight. Hybridization with DIG labelled probe and detection was done as per the protocol of Roche Applied Sciences.

 ~ Results Top

A total of 60 isolates, 18 drug resistant (including 12 MDRs), 18 drug sensitive and 24 mono drug resistant to either rifampicin (RIF) or isonicotinic acid hydrazide (INH) or other drugs (ciprofloxacin, ofloxacin, ethambutol) were included in the study. These 60 M.tuberculosis isolates were analyzed by IS 6110 DNA fingerprinting. The number of IS 6110 copies per isolate studied varied from 0 to 17 bands. The results of IS 6110 RFLP of 60 M.tuberculosis isolates can be classified into three groups namely A, B and C having either no copies, 1-4 copies or 5-17 copies of IS 6110, respectively. Of 60 M. tuberculosis isolates, 5 (8.3%) isolates belonged to group A, 8 (13.3%) isolates to group B and 47 (78.3%) isolates to group C.
Majority of isolates had multiple IS 6110 copies. Many isolates had DNA fingerprints showing some resemblance with each other but were not identical. Comparison of IS 6110 copy numbers and size profiles did not show any clustering among these isolates. All the three groups were found in drug resistant as well as in drug sensitive strains. The RFLP patterns obtained did not show any correlation with drug susceptibility or resistance. Isolates of patients living in same locality and having family contacts revealed different fingerprints.

 ~ Discussion Top

Identification and differentiation of strains of M.tuberculosis by restriction fragment length polymorphism (RFLP) of targeted insertion elements has provided a better understanding of the epidemiology of infection due to the pathogen in developed countries[4] and tracing the transmission.[6],[14] However, the situation is different in the developing countries like India as one of the countries with the largest burden of TB patients.[1] There are few studies on epidemiology of human tuberculosis using DNA fingerprinting of Indian strains of M.tuberculosis.[7],[8],[9],[10],[11] In the studies from south India, a large number of isolates of M. tuberculosis with low copy numbers or no copies of IS 6110 element have been observed.[7]
In the present study, with IS 6110 DNA fingerprinting, different hybridization patterns were observed for M.tuberculosis isolates from Agra, suggesting differences in copy number and genomic location of the element. The results obtained from this study demonstrate that the majority of isolates have multiple IS 6110 copies along with some few copy and zero copy numbers. Results show that there are different subpopulations of M.tuberculosis strains in Agra with distinct IS 6110 patterns. The first subpopulation is defined by strains that have zero copy numbers having no bands, the second is defined by strains that have low copy numbers, i.e., less than 4 bands and the third is defined by strains that have high copy numbers. This division agrees, by and large, with the observations of Yang et al with Asian isolates,[5] who noted the existence of an Asian subgroup of M. tuberculosis strains with low copy numbers. Our observations are in concordance with our previous studies from north Indian isolates which also revealed that the high percentage of multiple copies, and the RFLP patterns obtained, have no correlation with drug susceptibility or resistance.[10] While the trends in detecting low IS 6110 copy numbers are similar to most of Indian studies reported so far, the proportion of isolates with zero or few copies was comparatively lower than south Indian studies.[7],[9],[11]
Comparison of IS 6110 copy numbers and size profiles showed no clustering among these isolates. This could well be a reflection of the random study of small number of our isolates, since proper systemic family contact study could not be carried out. To study the epidemiology of tuberculosis, it is often important to establish the source of infection and to determine whether the disease is due to a new strain of M.tuberculosis or due to relapse of a single strain that is disseminating in a particular population. A recent study from south India using a combination of IS 6110 and direct repeat (DR) probes revealed that 38% shared a strain with other patients, indicating recent infection among older patients living in a community and those hospitalized in a sanatorium.[12]
Fingerprinting methods using IS 6110 will not be very useful in characterizing isolates with few or no copies of the element, and use of alternative methods of fingerprinting like DR probes, spoligotyping, AFLP, etc. are needed. To investigate molecular epidemiology among the isolates from Agra region, a study needs to be carried out on a larger number of samples from patients of defined localities and house holds for understanding the dynamics of infection.

 ~ Acknowledgement Top

We are grateful to Dr. SI Mehdi, formerly Director, TBDTC, Agra, for permitting to collect the clinical samples. Gift of reagent/plastic ware by LEPRA (UK) is gratefully acknowledged. 

 ~ References Top

1.World Health Organization (WHO). Global Tuberculosis Control. WHO Report 2001, Geneva: WHO, 2001.  Back to cited text no. 1    
2.Eisenach KD, Crawford JT, Bates JH. Repetitive DNA sequences as probes for Mycobacterium tuberculosis. J Clin Microbiol 1988;26:2240-2245.  Back to cited text no. 2    
3.van Embden JDA, Cave MD, Crawford JT, Dale J, Eisenach KD, Gicquel B, Hermans P, Martin C, McAdam R, Shinnikl TM, Small PM. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 1993;31:406-409.  Back to cited text no. 3    
4.van Soolingen D, de Hass PEW, Hermans PWM, Groenen PMA, van Embeden JDA. Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis. J Clin Microbiol 1993;31:1987-1995.  Back to cited text no. 4    
5.Yang ZH, Mtoni I, Mchonde, Mwasekaga M, Fuursted K, Askgard DS, Bennedson J, de Haas PEW, van Soolingen D, van Embden JDA, Anderson AB, DNA fingerprinting and phenotyping of Mycobacterium tuberculosis isolates from human immuno deficiency virus (HIV) seropositive and HIV - seronegative patients in Tanzania. J Clin Microbiol 1995;33:1064-1069.  Back to cited text no. 5    
6.Alland D, Kalkut GE, Moss AR, McAdam RA, Hahn JA, Bosworth W, Drucker E, Bloom BR. Transmission of tuberculosis in New York city analysis by DNA fingerprinting and conventional epidemiological methods. New Eng J Med 1994;330: 1710-1716.  Back to cited text no. 6    
7.Das S, Paramsivan CN, Lowrie DB, Prabhakar R, Narayanan PR. IS 6110 restriction fragment length polymorphism typing of clinical isolates of M.tuberculosis from patients with pulmonary tuberculosis in Madras, India. Tub Lung Dis 1995;76:550-554.  Back to cited text no. 7    
8.Narayanan S, Sahadevan R, Narayanan PR, Krishnamurthy PV, Paramsivan CN, Prabhakar R. RFLP of M.tuberculosis strain from various region of India using direct repeat probe. Indian J Med Res 1997;106:447-484.  Back to cited text no. 8    
9.Radhakrishnan I, Manju VK, Kumar AR, Mundayoor S. Implications of low frequency of IS 6110 in fingerprinting field isolates of M.tuberculosis from Kerala, India. J Clin Microbiol 2001;39:1683.  Back to cited text no. 9    
10.Siddiqi N, Shamim Md., Amin A, Chauhan DS, Das R, Srirvastava K, Sigh D, Sharma VD, Katoch VM, Sharma SK, Haneif M, Hasnain SE. Typing of drug resistant isolates of M.tuberculosis from India using the IS 6110 element reveals substantive polymorphism. Infect Genet Evol 2001;1:109-116.  Back to cited text no. 10    
11.Sahadevan R, Narayanan S, Paramsivan CN, Prabhakar R, Narayanan PR. Restriction fragment length polymorphism typing of clinical isolates of M.tuberculosis in Madras, India by use of direct repeat probe. J Clin Microbiol 1995;33:3037-3039.  Back to cited text no. 11    
12.Narayanan S, Das S, Garg R, Hari L, Rao VB, Frieden TR, Narayanan PR. Molecular epidemiology of tuberculosis in a rural area of high prevalence in south India: Implications for disease control and prevention. J Clin Microbiol 2002;40:4785-4788.  Back to cited text no. 12    
13.Vestal AL. In: Procedure for isolation and identification of mycobacteria, US department of Health, Education and Welfare, Publ.No. CDC 1977;77:8230 CDC Atlanta, Georgia.  Back to cited text no. 13    
14.Small PM, Shafer W, Hopewell PC, Singh SP, Murphy MJ, Desmond E, Sieraa M. Exogenous reinfection with multidrug resistant tuberculosis in patients with advanced HIV infection. N Eng J Med 1993;328:1137-1143.  Back to cited text no. 14    
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