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COMPARATIVE MIRU-VNTR PROFILE of the LCP TB ISOLATES and the WILD TYPE STRAIN H37Rv Crist John M. Pastor1, Ursela G. Bigol1, Jay T. Dalet1, Maria Teresa A. Barzaga2, Nelia S.Tan-Liu2 and Francisco M. Heralde III1 1Department

of Biochemistry and Molecular Biology, College of Medicine, University of the Philippines-Manila and 2 Lung Center of the Philippines, Quezon City, Philippines   Corresponding author: FM Heralde III, [email protected]

ABSTRACT The Mycobacterium Interspersed Repeating Units-Variable Number Tandem Repeats (MIRU-VNTR) profiling of heat killed Mycobacterium tuberculosis isolates from the Lung Center of the Philippines was done through optimization of the genotyping procedure using 12 MIRU-VNTR primers to amplify the MIRU-VNTRs of the wild type Mycobacterium tuberculosis strain, H37Rv. The PCR products were sequenced and verified through online NCBI GenBank BLAST tool. The optimized protocol was then used to amplify MIRU-VNTRs from the Lung Center of the Philippines and further calculate the number of MIRU-VNTR repeats which served as identification code for each isolate. Results show that some MIRU-VNTRs are highly polymorphic while others are moderately variable across strains. Comparison of the profile of the samples and with the wild type H37Rv show that different strains of Mycobacterium tuberculosis thrive in our country which calls for further studies on correlation of these observed differences on bacterial characteristics.

Methodology Optimization of MIRU - VNTR Amplification Process

INTRODUCTION

Tuberculosis

is one of the most deadly and widespread infectious diseases accounting for 2 to 3 million deaths worldwide1. According to the World Health Organization Global TB Report in 2006, one-third of the entire world’s population is infected with the disease2 and incidence is highest in Africa and Sub-Saharan regions. Among the South East Asian nations, the Philippines is considered as the ninth highest burden in terms of mortality as of 20083. Extensive efforts are being exerted by different countries to address this problem through drug development and improvement of diagnostic procedures, processes which are made complicated by emergence of drug-resistant strains that are less susceptible or even highly resistant to anti-tuberculosis antibiotics. Studies have shown that strains of Mycobacterium tuberculosis undergo mutation and there are existing predominant types of Mycobacterial strain that have developed stable association with their target human host population4. This emphasizes the point that specific strain of tuberculosis must first be identified and its characteristics are determined before administering treatment to prevent drug resistance. It can be done through genotyping using Variable Number of Tandem Repeats (VNTRs). This method relies on the length variation of twelve independent mini-satellite loci scattered through the Mycobacterium tuberculosis genome.7 MIRU or mycobacterial interspersed repeating units are VNTRs specific to mycobacteria and can be considered as the most practical and efficient method owing to its portability compared with other typing methods like IS6110, the “gold standard” in TB genotyping that requires highly trained personnel, sub-culturing of the bacteria and a turn around time of 30 days. MIRU-VNTR typing can also address the problem of low copy number of insertions which is a feature of highly infectious TB strain such as the MTb Beijing strain5. Since this technology is PCR-based, typing procedure can be done in less than 5 hours. The gel profile of the PCR products can be used to calculate the number of repeats for each VNTR locus and these set of numbers can be used as code for a particular isolate. This study aims to compare the MIRU profiles of TB isolates from the Lung Center of the Philippines (LCP) and compare them with the wild type tuberculosis strain, H37Rv.

REFERENCES 1. Burgos M.V. and Pym A.S., Molecular Epidemiology of Tuberculosis, European Respiratory Journal, pp. 54-60, doi: 10.183/09031936.02.00400702 2. WHO Global TB Report 2006, downloaded at the WHO website, www.WHO.com 3. WHO Global TB Control Report 2008, downloaded at the WHO website, www.WHO.com 4. Hirsh A.E., Tsolaki A.G., DeReimer K., Feldman M.W. and Small P.M., Stable Association Between the Strains of Mycobacterium tuberculosis and Their Human Host Populations, PNAS, 101(14), pp. 4871-4876 5. Spurgiesz S., Qitugua T., Smith K., Schupp J., Palmer E., Cox R and Keim P., “ Molecular Typing of Mycobacterium tuberculosisby Using Nine Novel Variable Number Tandem Repeats across the Beijing Family and Low-copy –Number IS6110 Isolates” Journal of Clinical Microbiology, September 2003 pp. 4224-4230 Vol.41 No.9 DOI 10.1128/JCM.41.9.4224-4230.2003. 6. Kam, K. M., Yip, C. W., Tse, L. W., Wong, K. L., Lam, T. K., Kremer, K., Au, B. K. & van Soolingen, D. (2005). Utility of mycobacterial interspersed repetitive unit typing for differentiating multidrugresistant Mycobacterium tuberculosis isolates of the Beijing family. J Clin Microbiol 43, 306–313. 7. Yong-Jiang Sun, Richard Bellamy, Ann S. G. Lee, Sze Ta Ng, Sindhu Ravindran, Sin-Yew Wong, Camille Locht, Philip Supply and Nicholas I. Paton (2204) Use of Mycobacterial Interspersed Repetitive Unit–Variable-Number Tandem Repeat Typing To Examine Genetic Diversity of Mycobacterium tuberculosis in Singapore. Journal of Clinical Microbiology Vol. 42, No. 5 DOI: 10.1128/JCM.42.5.1986–1993.2004

Acknowledgements The authors would like to thank University of the Philippines-Manila, the National Institutes of Health, Lung Center of the Philippines and MACARE Medicals for their continued support for the Mycobacterium tuberculosis Genotyping Project.

Mtb ( H37Rv) Bacteria Grown in Culture Medium

DNA Lysate Preparation

DNA Lysates from Lung Center of the Philippines

PCR Amplification

Separation by Gel Electrophoresis

OPTIMIZED MIRU - VNTR PROTOCOL

Visualization thru BioRad GelDoc System

Sequencing of PCR Products and NCBI BLAST Analysis

Calculation of MIRU - VNTR Repeats and Designation of Bacterial Identification Codes

RESULTS AND DISCUSSION The PCR results (Fig 1) show that ten out of twelve MIRUs were successfully amplified and visualized. Using linear regression, the formula for the number of base pairs for any band with a given distance travelled was calculated. The number of repeats of the following MIRUs was then computed through linear regression (see Table 1).     The sequences of the amplicons were determined from submitted PCR products to Macrogen, Korea. The results were verified through an online BLAST tool of NCBI GenBank. The ten MTB amplicons visualized in the gel  provided a correct match  on   the MIRU sequence of   the H37Rv gene. These findings suggest that the primers used as well as the PCR conditions utilized correctly determine the number of MIRUs of a given TB isolate . The optimized protocol was then used to determine the number of repeats for the other isolates obtained from LCP. The number of repeats obtained is shown in Table 2.

CONCLUSION

The number of repeats exhibited by the LCP isolates was different from the wild type H37Rv. Of interest is the apparent differential polymorphism of the MIRUs. Only MIRU 26 remained the same for the four isolates including the wild type strain. Majority of the MIRUs exhibited moderate polymorphism (MIRU 2, 4, 10, 16, 20, 24, 27, 31, 39 and 40). One MIRU exhibited high polymorphism (MIRU 23), suggestive of its strong potential in differentiating the local Tb isolates. Eleven out of 12 MIRUs were able to differentiate the wild type H37Rv from the LCP isolates. The same set of MIRUs was able to rule out the three LCP isolates to be different from each other. Notable is the difference obtained in MIRU 2, 4 and 26 in Table 1 and Table 2 for H37Rv. This difference in the computation of number of repeats will be further validated.

The optimized protocol in determining the number of MIRU-VNTRs has been validated by the congruence of the obtained MIRU sequences of H37Rv with those of the GenBank. The number of repeats found in the wild type varies from the other isolates obtained from LCP, proving that different strains of Mycobacterium tuberculosis thrive in our country. These differences could be correlated to differences in susceptibility to drugs and pathogenicity. As more LCP isolates will be tested in the future to comprise an MDRTB database, a phylogenetic analysis could be constructed from this MIRU profile to establish relationships and clustering of the different Tb strains.