IS6110 Transposition and Evolutionary Scenario of the Direct Repeat Locus in a Group of Closely Related Mycobacterium tuberculosis Strains

In recent years, various polymorphic loci and multicopy insertion elements have been discovered in the Mycobacterium tuberculosis genome, such as the direct repeat (DR) locus, the major polymorphic tandem repeats, the polymorphic GC-rich repetitive sequence, IS6110, and IS1081. These, especially IS6110 and the DR locus, have been widely used as genetic markers to differentiate M. tuberculosis isolates and will continue to be so used, due to the conserved nature of the genome of M. tuberculosis. However, little is known about the processes involved in generating these or of their relative rates of change. Without an understanding of the biological characteristics of these genetic markers, it is difficult to use them to their full extent for understanding the population genetics and epidemiology of M. tuberculosis. To address these points, we identified a cluster of 7 isolates in a collection of 101 clinical isolates and investigated them with various polymorphic genetic markers, which indicated that they were highly related to each other. This cluster provided a model system for the study of IS6110 transposition, evolution at the DR locus, and the effects of these on the determination of evolutionary relationships among M. tuberculosis strains. Our results suggest that IS6110 restriction fragment length polymorphism patterns are useful in grouping closely related isolates together; however, they can be misleading if used for making inferences about the evolutionary relationships between closely related isolates. DNA sequence analysis of the DR loci of these isolates revealed an evolutionary scenario, which, complemented with the information from IS6110, allowed a reconstruction of the evolutionary steps and relationships among these closely related isolates. Loss of the IS6110 copy in the DR locus was noted, and the mechanisms of this loss are discussed.     

Genetic Diversity of Mycobacterium tuberculosis from Guadalajara,Mexico and Identification of a Rare Multidrug Resistant Beijing Genotype

Determining the genetic diversity of M. tuberculosis strains allows identification of the distinct Mycobacterium tuberculosis genotypes responsible for tuberculosis in different regions. Several studies have reported the genetic diversity of M. tuberculosis strains in Mexico, but little information is available from the state of Jalisco. Therefore, the aim of this study was to determine the genetic diversity of Mycobacterium tuberculosis clinical isolates from Western Mexico. Sixty-eight M. tuberculosis isolates were tested for susceptibility to first-line drugs using manual Mycobacteria Growth Indicator Tube method and genotyped using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) pattern analyses. Forty-seven (69.1%) isolates were grouped into 10 clusters and 21 isolates displayed single patterns by spoligotyping. Three of the 21 single patterns corresponded to orphan patterns in the SITVITWEB database, and 1 new type that contained 2 isolates was created. The most prevalent lineages were T (38.2%), Haarlem (17.7%), LAM (17.7%), X (7.4%), S (5.9%), EAI (1.5%) and Beijing (1.5%). Six (12.8%) of the clustered isolates were MDR, and type 406 of the Beijing family was among the MDR isolates. Seventeen (26.2%) isolates were grouped into 8 clusters and 48 isolates displayed single patterns by IS6110-RFLP. Combination of IS6110-RFLP and spoligotyping reduced the clustering rate to 20.0%. The results show that T, Haarlem, and LAM are predominant lineages among clinical isolates of M. tuberculosis in Guadalajara, Mexico. Clustering rates indicated low transmission of MDR strains. We detected a rare Beijing genotype, SIT406, which was a highly resistant strain. This is the first report of this Beijing genotype in Latin America.     

Transposition mechanism,molecular characterization and evolution of IS<Emphasis Type="Italic">6110</Emphasis>, the specific evolutionary marker of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> complex

The mycobacterial insertion sequence IS6110 proved crucial in deciphering tuberculosis (TB) transmission dynamics. This sequence was also shown to play an important role in the pathogenicity (transmission ability and/or virulence) of Mycobacterium tuberculosis, the main causative agent of TB in humans. In this study, we explored the usefulness of IS6110 and its potential as a phylogenetic/typing marker. We also analyzed the genetic polymorphism and evolutionary trends (selective pressure) of its transposase-encoding open reading frames (ORFs), A and B, using the maximum likelihood method. Both ORFs evolved chronologically through random single nucleotide polymorphisms. They were subjected to strict purifying selection more tight on orfA, with no evidence of significant recombination events. OrfA proved to have a crucial role in regulating the transpositional process. Several analyses showed that IS6110 acquisition antedated the emergence of the Mycobacterium tuberculosis complex. This original copy of IS6110 element was functionally optimal. In conclusion, this study not only demonstrated the usefulness of IS6110 in terms of phylogenetic and typing purposes and its transpositional mechanism, but also informed the scientific community on its evolutionary history.     

Evolutionary Trends of the Transposase-Encoding Open Reading Frames A and B (orfA and orfB) of the Mycobacterial IS6110 Insertion Sequence

Background

The IS6110 insertion sequence, a member of the IS3 family of insertion sequences, was found to be specific to the Mycobacterium tuberculosis complex (MTBC). Although IS6110 has been extensively characterized as a transposable genetic marker, the evolutionary history of its own transposase-encoding sequence has not, to the best of our knowledge, been investigated.

Methodology/Principal Findings

Here we explored the evolution of the IS6110 sequence by analysing the genetic variability and the selective forces acting on its transposase-encoding open reading frames (ORFs) A and B (orfA and orfB). For this purpose, we used a strain collection consisting of smooth tubercle bacilli (STB), an early branching lineage of the MTBC, and present-day M. tuberculosis strains representing the full breadth of genetic diversity in Tunisia. In each ORF, we found a major haplotype that dominated over a flat distribution of rare descendent haplotypes, consisting mainly of single- and double-nucleotide variant singletons. The predominant haplotypes consisted of both ancestral and present-day strains, suggesting that IS6110 acquisition predated the emergence of the MTBC. There was no evidence of recombination and both ORFs were subjected to strict purifying selection, as demonstrated by their dN/dS ratios (0.29 and 0.51, respectively), as well as their significantly negative Tajima’s D statistics. Strikingly, the purifying selection acting on orfA proved much more stringent, suggesting its critical role in regulating the transpositional process. Maximum likelihood analyses further excluded any possibility of positive selection acting on single amino acid residues.

Conclusions/Significance

Taken together our data fit with an evolutionary scenario according to which the observed variability pattern of the IS6110 transposase-encoding ORFs is generated mainly through random point mutations that accrued on a functionally optimal IS6110 copy, whose acquisition predated the emergence of the MTBC complex. Background selection acting against deleterious mutations led to an excess of low-frequency variants.     

Rapid detection of Mycobacterium tuberculosis in clinical samples by multiplex polymerase chain reaction (mPCR)

Although the multi-copy and specific element IS6110 provides a good target for the detection of Mycobacterium tuberculosis complex by PCR techniques, the emergence of IS6110-negative strains suggested that false negative may occur if IS6110 alone is used as the target for detection. In this report, a multiplex polymerase chain reaction (mPCR) system was developed using primers derived from the insertion sequence IS6110 and an IS-like elements designated as B9 (GenBank accession no. U78639.1) to overcome the problem of detecting negative or low copy IS6110 containing strains of M. tuberculosis. The mPCR was evaluated using 346 clinical samples which included 283 sputum, 19 bronchial wash, 18 pleural fluid, 9 urine, 7 CSF, 6 pus, and 4 gastric lavage samples. Our results showed that the sensitivity (93.1 %) and specificity (89.6 %) of the mPCR system exceeds that of the conventional method of microscopy and culture. The mPCR assay provides an efficient strategy to detect and identify M. tuberculosis from clinical samples and enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.     

Insertion sequence-caused large-scale rearrangements in the genome of Escherichia coli

A majority of large-scale bacterial genome rearrangements involve mobile genetic elements such as insertion sequence (IS) elements. Here we report novel insertions and excisions of IS elements and recombination between homologous IS elements identified in a large collection of Escherichia coli mutation accumulation lines by analysis of whole genome shotgun sequencing data. Based on 857 identified events (758 IS insertions, 98 recombinations and 1 excision), we estimate that the rate of IS insertion is 3.5 × 10⁻⁴ insertions per genome per generation and the rate of IS homologous recombination is 4.5 × 10⁻⁵ recombinations per genome per generation. These events are mostly contributed by the IS elements IS1, IS2, IS5 and IS186. Spatial analysis of new insertions suggest that transposition is biased to proximal insertions, and the length spectrum of IS-caused deletions is largely explained by local hopping. For any of the ISs studied there is no region of the circular genome that is favored or disfavored for new insertions but there are notable hotspots for deletions. Some elements have preferences for non-coding sequence or for the beginning and end of coding regions, largely explained by target site motifs. Interestingly, transposition and deletion rates remain constant across the wild-type and 12 mutant E. coli lines, each deficient in a distinct DNA repair pathway. Finally, we characterized the target sites of four IS families, confirming previous results and characterizing a highly specific pattern at IS186 target-sites, 5′-GGGG(N6/N7)CCCC-3′. We also detected 48 long deletions not involving IS elements.     

Clinical evaluation of mtp40 polymerase chain reaction for the diagnosis of extra pulmonary tuberculosis

Rapid and sensitive detection of Mycobacterium tuberculosis from patient samples is vital for clinical diagnosis and treatment. The emergence of M. tuberculosis strains with either no copies or only a single copy of IS6110 in Asian countries makes the standard PCR based diagnosis of M. tuberculosis using IS6110 not reliable. We studied the diagnostic efficacy of the in-house PCR amplification of the candidate gene mtp40 as an alternative to IS6110 element based diagnosis. Clinical samples included pulmonary and extra-pulmonary specimens from TB suspected patients residing in Puducherry, South India and were analyzed using in-house PCR procedures targeting IS6110 element and mtp40 genes. Out of 317 clinical specimens analyzed, 132 (41.6 %) and 114 (36 %) were found positive for mtp40 PCR and IS6110 PCR, respectively. However, 18 specimens that were found to negative for IS6110 PCR were found positive for mtp40 PCR, which was further confirmed by DNA sequencing method. PCR amplification of mtp40 gene for the diagnosis of M. tuberculosis in clinical samples is fast, sensitive, and further identified clinical strains that lack IS6110 element in this region. It is clearly demonstrated that there is a significant difference between the two PCR procedures and the sensitivity and specificity levels of mtp40 PCR were found to be higher when compared with DNA sequencing method. Thus, mtp40 based PCR technique will be beneficial in diagnosis of TB where M. tuberculosis strains lack of IS6110 element is predominant.     

Simplified amplified-fragment length polymorphism method for genotyping Mycobacterium tuberculosis isolates

A simplified amplified-fragment length polymorphism (AFLP) method was developed and applied to genotype 52 Mycobacterium tuberculosis isolates. This method can be carried out using only one restriction enzyme (XhoI), one double strand adapter, and one PCR primer. The amounts of DNA and DNA polymerase, and the concentrations of primer and Mg²⁺ in the PCR step were optimized using the Basic Sequential Simplex method. AFLP analysis of the isolates generated a total of 24 differently sized bands ranging from 1537 to 121 bp, and 52 different band patterns, with a minimum of 2 and a maximum of 13 bands. The results were compared with the well-established IS6110 restriction fragment length polymorphism (IS6110-RFLP) typing method, which rendered a total of 32 differently sized bands from 1 to 12 kbp, and 52 different band patterns, with a minimum of 3 and a maximum of 15 bands. Therefore, both genotyping methods showed a discriminatory power of samples of 100%. Nevertheless, pairwise comparisons of the 1326 similarity indexes calculated for both typing methods showed a total absence of correlation between the similarity indexes of the two methods. The simplified AFLP method is expected to be more useful for genotyping M. tuberculosis isolates compared to the IS6110-RFLP method, since the former evaluates genetic variations throughout the M. tuberculosis genome. Furthermore, the relatively rapid and low-cost simplified AFLP method compares favorably to the IS6110-RFLP or conventional AFLP methods, and shows great promise for genotyping M. tuberculosis isolates, especially in developing countries or for preliminary screening.     

Studies of Polymorphic DNA Fingerprinting and Lipid Pattern of Mycobacterium tuberculosis Patient Isolates in Japan

Strain differentiation by DNA restriction fragment length polymorphism (RFLP) has been used mainly for the epidemiological purpose of Mycobacterium tuberculosis infection. In this study, we tried to connect the molecular and phenotypic characteristics of M. tuberculosis patient isolates by comparing the DNA fingerprints obtained by RFLP using IS6110 and lipid patterns using two-dimensional thin-layer chromatography (2-D TLC) with silica gel, since M. tuberculosis has a lipid-rich cell envelope which contributes to the virulence and immunomodulatory properties. We found that 66 isolates of M. tuberculosis from tuberculosis patients showed that the occurrence of IS6110 varied from 1 to 24 copies. The IS6110 patterns were highly variable among isolates. Fifty different RFLP patterns were observed, and 12 RFLP patterns were shared by two or more strains. By computerized analysis of the RFLP patterns of M. tuberculosis patient isolates, we found that 95% of the isolates fell into seven clusters, from A to G, with at least two isolates in each (> 30% similarity). Among the cellular lipids, the phospholipid composition did not differ by strain, whereas the glycolipid pattern differed markedly. Especially, the relative concentration of cord factor and sulfolipid, both of which were known as virulent factors, varied by strain. The fingerprints of some strains showed an association between the DNA and glycolipid patterns, even though some of the same DNA fingerprint strains showed differences in lipid patterns. Among the patient isolates, M. tuberculosis strain 249 possessed a specific glycolipid with 2-O-methyl-L -rhamnose and L-rhamnose, which is rarely found in other strains. This glycolipid showed serological activity against the sera of tuberculosis patients, even if the reactivity was not as strong as trehalose dimycolate. It also showed the inhibition of phagosome-lysosome fusion in macrophages, suggesting involvement with virulence. These results suggest that RFLP analysis using IS6110 is useful for clustering the human isolates of M. tuberculosis, however, for further strain differentiation on virulence, a lipid analysis provides more information.     

An efficient alternative marker for specific identification of Mycobacterium tuberculosis

Rapid and accurate identification of mycobacteria to the species level is important to provide epidemiological information and to guide the appropriate treatment, especially identification of the Mycobacterium tuberculosis (MTB) which is the leading pathogen causing tuberculosis. The genetic marker named as Mycobacterium tuberculosis specific sequence 90 (mtss90) was screened by a bioinformatics software and verified by a series of experiments. To test its specificity, 266 strains of microorganisms and human cells were used for the mtss90 conventional PCR method. Moreover, the efficiency of mtss90 was evaluated by comparing 16S rDNA (Mycobacterium genus-specific), IS6110 (specific identification of MTB complex), mtp40 (MTB-specific) and PNB/TCH method (traditional bacteriology testing) in Mycobacterium strains. All MTB isolates were mtss90 positive. No amplification was observed from any other tested strains with M. microti as an exception. Compared with the traditional PNB/TCH method, the coincidence rate was 99.1 % (233/235). All of the mtss90 positive strains were IS6110 and 16S rDNA positive, indicating a 100 % coincidence rate (216/216) between mtss90 and these two genetic markers. Additionally, mtss90 had a better specificity than mtp40 in the identification of MTB. Lastly, a real-time PCR diagnostic assay was developed for the rapid identification of MTB. In conclusion, mtss90 may be an efficient alternative marker for species-specific identification of MTB and could be used for the diagnosis of tuberculosis combined with other genetic markers.     

Involvement of transposable elements in the formation of hybrid phages between bacteriophage P1 and the R plasmid NR1

Homologous recombination between IS1 elements present on both replicons, P1 and NR1, resulted in P1-NR1 cointegrates and P1-RTF and P1-r-det phages. Cointegration between P1 and NR1-B, and NR1 derivative with multiple DNA rearrangements including insertion of the transposable element γδ, was also mediated by reciprocal recombination in IS1 sequences. However, all 4 hybrids studied carried deletions promoted by γδ residing on NR1-B. Further IS1-mediated deletions on the hybrid genomes resulted in plaque-forming P1Cm phages.     

Distribution of spoligotyping defined genotypic lineages among drug-resistant Mycobacterium tuberculosis complex clinical isolates in Ankara, Turkey

Background

Investigation of genetic heterogeneity and spoligotype-defined lineages of drug-resistant Mycobacterium tuberculosis clinical isolates collected during a three-year period in two university hospitals and National Tuberculosis Reference and Research Laboratory in Ankara, Turkey.

Methods and Findings

A total of 95 drug-resistant M. tuberculosis isolates collected from three different centers were included in this study. Susceptibility testing of the isolates to four major antituberculous drugs was performed using proportion method on Löwenstein–Jensen medium and BACTEC 460-TB system. All clinical isolates were typed by using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) methods. Seventy-three of the 95 (76.8%) drug resistant M. tuberculosis isolates were isoniazid-resistant, 45 (47.4%) were rifampicin-resistant, 32 (33.7%) were streptomycin-resistant and 31 (32.6%) were ethambutol-resistant. The proportion of multidrug-resistant isolates (MDR) was 42.1%. By using spoligotyping, 35 distinct patterns were observed; 75 clinical isolates were grouped in 15 clusters (clustering rate of 79%) and 20 isolates displayed unique patterns. Five of these 20 unique patterns corresponded to orphan patterns in the SITVIT2 database, while 4 shared types containing 8 isolates were newly created. The most prevalent M. tuberculosis lineages were: Haarlem (23/95, 24.2%), ill-defined T superfamily (22/95, 23.2%), the Turkey family (19/95, 20%; previously designated as LAM7-TUR), Beijing (6/95, 6.3%), and Latin-America & Mediterranean (LAM, 5/95 or 5.3%), followed by Manu (3/95, 3.2%) and S (1/95, 1%) lineages. Four of the six Beijing family isolates (66.7%) were MDR. A combination of IS6110-RFLP and spoligotyping reduced the clustering rate from 79% to 11.5% among the drug resistant isolates.

Conclusions

The results obtained showed that ill-defined T, Haarlem, the Turkey family (previously designated as LAM7-TUR family with high phylogeographical specifity for Turkey), Beijing and LAM were predominant lineages observed in almost 80% of the drug-Resistant M. tuberculosis complex clinical isolates in Ankara, Turkey.     

Drug resistance and IS6110‐RFLP patterns of Mycobacterium tuberculosis in patients with recurrent tuberculosis in northern Thailand

The emergence of drug resistant Mycobacterium tuberculosis has become a global threat to tuberculosis (TB) prevention and control efforts. This study aimed to determine the drug resistance profiles and DNA fingerprints of M. tuberculosis strains isolated from patients with relapsed or retreatment pulmonary TB in Chiang Rai province in northern Thailand. Significant differences in multidrug resistance (MDR) (P = 0.025) and resistance to isoniazid (P = 0.025) and rifampin (P = 0.046) between first and second registrations of patients with retreatment TB were found. However, there were no significant differences in resistance to any drugs in patients with relapsed TB. The rate of MDR‐TB strains was 12.2% among new patients at first registration, 22.5% among patients with recurrence who had previously undergone treatment at second registration and 12.5% at third registration. Two retreatment patients whose initial treatment had failed had developed MDR‐TB with resistance to all TB drugs tested, including rifampin, isoniazid, streptomycin and ethambutol. IS6110‐RFLP analysis revealed that 66.7% (10/15 isolates) of MDR‐TB belonged to the Beijing family. In most cases, IS6110‐RFLP patterns of isolates from the same patients were identical in relapse and retreatment groups. However, some pairs of isolates from retreatment patients after treatment failure had non‐identical IS6110‐RFLP patterns. These results suggest that, after failure and default treatment, patients with retreatment tuberculosis have a significantly greater risk of MDR‐TB, isoniazid and rifampin resistance than do other patients.     

Evolution of rhizobial symbiosis islands through insertion sequence-mediated deletion and duplication

Symbiosis between organisms influences their evolution via adaptive changes in genome architectures. Immunity of soybean carrying the Rj2 allele is triggered by NopP (type III secretion system [T3SS]-dependent effector), encoded by symbiosis island A (SymA) in B. diazoefficiens USDA122. This immunity was overcome by many mutants with large SymA deletions that encompassed T3SS (rhc) and N₂ fixation (nif) genes and were bounded by insertion sequence (IS) copies in direct orientation, indicating homologous recombination between ISs. Similar deletion events were observed in B. diazoefficiens USDA110 and B. japonicum J5. When we cultured a USDA122 strain with a marker gene sacB inserted into the rhc gene cluster, most sucrose-resistant mutants had deletions in nif/rhc gene clusters, similar to the mutants above. Some deletion mutants were unique to the sacB system and showed lower competitive nodulation capability, indicating that IS-mediated deletions occurred during free-living growth and the host plants selected the mutants. Among 63 natural bradyrhizobial isolates, 2 possessed long duplications (261–357 kb) harboring nif/rhc gene clusters between IS copies in direct orientation via homologous recombination. Therefore, the structures of symbiosis islands are in a state of flux via IS-mediated duplications and deletions during rhizobial saprophytic growth, and host plants select mutualistic variants from the resultant pools of rhizobial populations. Our results demonstrate that homologous recombination between direct IS copies provides a natural mechanism generating deletions and duplications on symbiosis islands.Subject terms: Soil microbiology, Molecular evolution     

Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method

Mycobacterium tuberculosis complex strains contain a unique chromosomal region, which consists of multiple 36bp direct repeats (DRs), which are interspersed by unique spacers 35 to 41 bp in length. In this study we investigated the nature of the DNA polymorphism of this DR cluster by sequencing part of this region in a large number of M. tuberculosis complex strains. Two types of genetic rearrangements were observed. One type consists of the variation in one or a few discrete, contiguous DRs plus spacer sequences. This variation is probably driven by homologous recombination between adjacent or distant DRs. The other type of polymorphism is probably driven by transpositional events of the insertion sequence, IS6110, which is almost invariably present in the DR cluster of M. tuberculosis complex strains. Based on the nature of the DNA polymorphism in the DR cluster, we developed a novel method of strain differentiation, direct variable repeat polymer chain reaction (DVR-PCR), which enables typing of individual M. tuberculosis strains in a single PCR. The method allows an excellent differentiation of epidemiologically unrelated isolates and, in principle, the DVR-PCR allows the detection of M. tuberculosis and strain differentiation at the same time.     

IS6110-mediated deletion polymorphism in the direct repeat region of clinical isolates of Mycobacterium tuberculosis

This study investigates the phenomenon of IS6110-mediated deletion polymorphism in the direct repeat (DR) region of the genome of Mycobacterium tuberculosis. Clinical isolates and their putative predecessors were compared using a combination of DR region restriction fragment length polymorphism, IS6110 DNA fingerprinting, spoligotyping, and DNA sequencing, which allowed the mapping of chromosome structure and deletion junctions. The data suggest that adjacently situated IS6110 elements mediate genome deletion. However, in contrast to previous reports, deletions appear to be mediated by inversely oriented IS6110 elements. This suggests that these events may occur via mechanisms other than RecA-mediated homologous recombination. The results underscore the important role of IS6110-associated deletion hypervariability in driving M. tuberculosis genome evolution.     

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein^1,2. The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale^1-4.Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).     

Comparison between RFLP and MIRU-VNTR Genotyping of Mycobacterium tuberculosis Strains Isolated in Stockholm 2009 to 2011

Our aim was to analyze the difference between methods for genotyping of Mycobacterium tuberculosis complex isolates. We collected genotyping results from Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetitive Units - Variable Numbers of Tandem Repeat (MIRU-VNTR) in a geographically limited area (Stockholm) during a period of three years. The number and proportion of isolates belonging to clusters was reduced by 45 and 35% respectively when combining the two methods compared with using RFLP or MIRU-VNTR only. The mean size of the clusters was smaller when combining methods and smaller with RFLP compared to MIRU-VNTR. In clusters with confirmed epidemiological links RFLP coincided slightly better than MIRU-VNTR but where there was a difference, the variation in MIRU-VNTR pattern was only in a single locus. In isolates with few IS6110 bands in RFLP, MIRU-VNTR differentiated the isolates more, dividing the RFLP clusters. Since MIRU-VNTR is faster and less labour-intensive it is the method of choice for routine genotyping. In most cases it will be sufficient for epidemiological purposes but true clustering might still be considered if there are epidemiological links and the MIRU-VNTR results differ in only one of its 24 loci.     

IS481 and IS1002 of Bordetella pertussis Create a 6-Base-Pair Duplication upon Insertion at a Consensus Target Site

The insertion sequence IS481 and its isoform IS1002 have been observed to transpose into the bvgAS locus of Bordetella pertussis, for which the DNA sequence has previously been determined. Upon insertion of IS481 at three different sites and IS1002 at one site, a 6-bp sequence originally present was found at the junction of bvg and insertion sequence DNA. This indicates that, contrary to prior reports, IS481 and IS1002 do create a duplication upon insertion. In this light, examination of these and other examples of IS481 and IS1002 reported in the literature leads to the observation that the 6-bp recognition sequence usually fits the consensus NCTAGN. The near-palindromic nature of this sequence, when directly repeated at the ends of IS481 or IS1002, apparently led to the interpretation that 5 of these base pairs were part of the terminal inverted repeats flanking these elements.