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Chapter 3 : Molecular Epidemiology of

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Abstract:

Epidemiologic investigations were limited because there are relatively few phage types, the most common of which are shared by many isolates, and antibiograms are useful only for drug-resistant strains. The modern genotyping methods and the relevance of these methods to the control and understanding of the pathogenesis of tuberculosis (TB) have recently been reviewed. Southern blots of DNA electrophoresed on agarose gels and probed with a fragment of IS that lies upstream of the single PvuII site provide restriction fragment length polymorphism (RFLP) patterns. Polymorphisms in the direct-repeat (DR) locus tend to group strains into larger groups than does Insertion sequence (IS) analysis and have been used to link strains to specific geographic areas. Polymorphisms in tandemly repeated minisatellite loci caused by unequal crossing over are the basis for human forensic DNA typing. Recently, several groups have used synonymous single-nucleotide polymorphisms (SNPs) as a tool to type strains. Point mutations are categorized as either nonsynonymous or synonymous, depending on whether the substitution leads to a change in amino acid sequence and thus in the protein for which the sequence codes. Genotyping of suspected false-positive cultures plays an important role in confirming false-positive cases and possible sources of cross-contamination. In a recent investigation in the United States, DNA fingerprinting was used to evaluate epidemiologically linked pairs found during contact investigations. The fact that strain W was originally associated with an outbreak of multidrug-resistant TB (MDRTB) confounds the issue of transmissibility/virulence and drug resistance.

Citation: Cave M, Murray M, Nardell E. 2005. Molecular Epidemiology of , p 33-46. In Cole S, Eisenach K, McMurray D, Jacobs, Jr. W (ed), Tuberculosis and the Tubercle Bacillus. ASM Press, Washington, DC. doi: 10.1128/9781555817657.ch3

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Figure 1

Chromosome of hypothetical strain X and genotyping of BCG, the laboratory strain H37Rv, and strain X on the basis of ISinsertion sites and MIRUs. The top right-hand panel shows the chromosome of hypothetical strain X. sites are indicated by the arrows. The top left-hand panel shows the results of IS-based genotyping. Mycobacterial DNA is digested with the restriction enzyme . The ISprobe hybridizes to ISDNA to the right of the site in IS. The size of each hybridizing fragment depends on the distance from this site to the next site in the adjacent DNA (fragments a through f), as reflected by gel electrophoresis of the DNA fragments of BCG, H37Rv, and X. The horizontal lines to the right of the electrophoretic strip indicate the extent of the distribution of fragments in the gel, including fragments that contain no IS. The three bottom panels show the results of MIRU-based genotyping. MIRUs contain repeat units, and MIRU analysis involves the use of PCR amplification and gel electrophoresis to categorize the number and size of the repeats at 12 independent loci, each of which has a unique repeated sequence. The sizes of molecular weight markers (lanes M) and PCR products for loci A, B, C, and D (lanes A to D, respectively), in BCG, H37Rv, and X are shown. The specific sizes of the various MIRUs in each strain result in a distinctive fingerprint for the strain. (Modified from reference 4 with permission. Copyright © 2003 Massachusetts Medical Society. All rights reserved.)

Citation: Cave M, Murray M, Nardell E. 2005. Molecular Epidemiology of , p 33-46. In Cole S, Eisenach K, McMurray D, Jacobs, Jr. W (ed), Tuberculosis and the Tubercle Bacillus. ASM Press, Washington, DC. doi: 10.1128/9781555817657.ch3
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Image of Figure 2
Figure 2

Spoligotyping. The DR locus is a chromosomal region that contains 10 to 50 copies of a 36-bp direct repeat, separated by spacer DNA with various sequences, each of which is 37 to 41 bp. A copy of ISis inserted within a 36-bp DR in the middle of the DR locus in most strains. strains have the same overall arrangement of spacers but differ in terms of the presence or absence of specific spacers. Spoligotyping involves PCR amplification of the DR locus, followed by hybridization of the labeled PCR products to a membrane that contains covalently bound oligonucleotides corresponding to each of 43 spacers. Individual strains have positive or negative signals for each spacer. The top section shows the 43 DR (rectangles) and spacers (horizontal lines) used in spoligotyping. The middle section shows the products of PCR amplification of spacers 1 through 6 in BCG, strain H37Rv, and hypothetical strain X, with the use of primers (white and black arrows) at each end of the DR loci. The bottom section shows the spoligotypes of the three strains. (Reprinted from reference 4 with permission. Copyright © 2003 Massachusetts Medical Society. All rights reserved.)

Citation: Cave M, Murray M, Nardell E. 2005. Molecular Epidemiology of , p 33-46. In Cole S, Eisenach K, McMurray D, Jacobs, Jr. W (ed), Tuberculosis and the Tubercle Bacillus. ASM Press, Washington, DC. doi: 10.1128/9781555817657.ch3
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