Use of multiple‐locus variable number tandem repeat analysis and phage typing for subtyping ofSalmonella Enteritidis from sporadic human cases in the United States

Aims: To investigate the genetic diversity among S. Enteritidis isolates from different geographic regions to evaluate the relationship between phage types (PTs) and variable number tandem repeat analysis (VNTR) loci. Methods and Results: We performed multiple‐locus variable number tandem repeat analysis (MLVA) and phage typing on 245 S. Enteritidis isolates collected from sporadic human clinical cases in Michigan, Minnesota, New York, and Washington states between 2000 and 2007. Ninety‐four MLVA types and 22 different PTs were identified. Specific PTs were associated with a predominant allele for certain VNTR loci. Cluster analysis using a minimum‐spanning tree demonstrated two major clusters (I, II) and one minor cluster of isolates. PTs 8, 13a, 13 and 34 were significantly associated with MLVA cluster I. Phage types 1, 4, 6a, and 18 were significantly associated with MLVA cluster II. Conclusions: We found significant association between MLVA‐based clusters and PTs. Certain VNTR loci were associated with specific PTs and could serve as useful molecular markers for S. Enteritidis in epidemiological investigations. Significance and Impact of the Study: MLVA genotyping in combination with phage typing can be used for effective characterization of S. Enteritidis isolates. It can also be useful for tracing possible sources during investigations of sporadic and outbreak cases of S. Enteritidis.     

DNA fingerprinting of Mycobacterium leprae strains using variable number tandem repeat (VNTR) - fragment length analysis (FLA)

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO, leprosy remains endemic in many countries with approximately 250,000 new cases each year. The entire M. leprae genome has been mapped and many loci have been identified that have repeated segments of 2 or more base pairs (called micro- and minisatellites). Clinical strains of M. leprae may vary in the number of tandem repeated segments (short tandem repeats, STR) at many of these loci. Variable number tandem repeat (VNTR) analysis has been used to distinguish different strains of the leprosy bacilli. Some of the loci appear to be more stable than others, showing less variation in repeat numbers, while others seem to change more rapidly, sometimes in the same patient. While the variability of certain VNTRs has brought up questions regarding their suitability for strain typing, the emerging data suggest that analyzing multiple loci, which are diverse in their stability, can be used as a valuable epidemiological tool. Multiple locus VNTR analysis (MLVA) has been used to study leprosy evolution and transmission in several countries including China, Malawi, the Philippines, and Brazil. MLVA involves multiple steps. First, bacterial DNA is extracted along with host tissue DNA from clinical biopsies or slit skin smears (SSS). The desired loci are then amplified from the extracted DNA via polymerase chain reaction (PCR). Fluorescently-labeled primers for 4-5 different loci are used per reaction, with 18 loci being amplified in a total of four reactions. The PCR products may be subjected to agarose gel electrophoresis to verify the presence of the desired DNA segments, and then submitted for fluorescent fragment length analysis (FLA) using capillary electrophoresis. DNA from armadillo passaged bacteria with a known number of repeat copies for each locus is used as a positive control. The FLA chromatograms are then examined using Peak Scanner software and fragment length is converted to number of VNTR copies (allele). Finally, the VNTR haplotypes are analyzed for patterns, and when combined with patient clinical data can be used to track distribution of strain types.     

Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica)

We studied inter-simple sequence repeat (ISSR) polymorphism and inheritance in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and sugi (Cryptomeria japonica D. Don) megagametophytes using primers that anneal to simple repeats of various lengths, sequences, and non-repetitive motifs at the 5 and 3 ends. Products were visualized on agarose gels with ethidium bromide staining. More than 60% of the 96 primers tested gave interpretable banding patterns in both Douglas-fir and sugi, and the useful primers were in complete agreement among species. Dinucleotide repeat primers were the majority of those tested, and gave all of the useful banding patterns. The 24 best primers were used for segregation studies, yielding a total of 77 loci distributed among two Douglas-fir families and one sugi family. Approximately 90% of the 24 primers showed polymorphism within at least one of the three families. The average number of variable loci per primer was 1.6. Primers based on (AG) _n repeats gave the largest number of polymorphic loci; 16 primer-family combinations yielded 24 segregating loci. However, primer based on (GT) _n repeats gave the most loci per primer studied (mean of 2.0). All markers displayed apparent dominance (band presence vs absence), and all but three segregation ratios (4%) fit Mendelian expectations: Because they employ longer primers than do RAPDs, have a high degree of polymorphism, conform well to Mendelian expectations, and do not require use of acrylamide gels for analysis, ISSRs may be useful markers for PCR-based genome maps and population studies of conifers.Paper 3082 of the Forest Research Laboratory, Oregon State University     

Characterization of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Central Asian Strain1 using mycobacterial interspersed repetitive unit genotyping

Background  

The Central Asian Strain1 (CAS1) genogroup of Mycobacterium tuberculosis (MTB) is the most prevalent in Pakistan, India and Bangladesh. Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing is a reliable and reproducible method for differentiation of MTB isolates. However, information of its utility in determining the diversity of CAS1 strain is limited. We performed standard 12 loci based MIRU-VNTR typing on previously spoligotyped CAS1 strains and 'unique' strains in order to evaluate its discriminatory power for these isolates.     

Genomic Diversity and Evolution of Mycobacterium ulcerans Revealed by Next-Generation Sequencing

Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease after tuberculosis and leprosy. It is an emerging infectious disease that afflicts mainly children and youths in West Africa. Little is known about the evolution and transmission mode of M. ulcerans, partially due to the lack of known genetic polymorphisms among isolates, limiting the application of genetic epidemiology. To systematically profile single nucleotide polymorphisms (SNPs), we sequenced the genomes of three M. ulcerans strains using 454 and Solexa technologies. Comparison with the reference genome of the Ghanaian classical lineage isolate Agy99 revealed 26,564 SNPs in a Japanese strain representing the ancestral lineage. Only 173 SNPs were found when comparing Agy99 with two other Ghanaian isolates, which belong to the two other types previously distinguished in Ghana by variable number tandem repeat typing. We further analyzed a collection of Ghanaian strains using the SNPs discovered. With 68 SNP loci, we were able to differentiate 54 strains into 13 distinct SNP haplotypes. The average SNP nucleotide diversity was low (average 0.06–0.09 across 68 SNP loci), and 96% of the SNP locus pairs were in complete linkage disequilibrium. We estimated that the divergence of the M. ulcerans Ghanaian clade from the Japanese strain occurred 394 to 529 thousand years ago. The Ghanaian subtypes diverged about 1000 to 3000 years ago, or even much more recently, because we found evidence that they evolved significantly faster than average. Our results offer significant insight into the evolution of M. ulcerans and provide a comprehensive report on genetic diversity within a highly clonal M. ulcerans population from a Buruli ulcer endemic region, which can facilitate further epidemiological studies of this pathogen through the development of high-resolution tools.     

Detection and Strain Typing of Ancient Mycobacterium leprae from a Medieval Leprosy Hospital

Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP) in Winchester, UK, showing skeletal signs of lepromatous leprosy (LL) have been studied using a multidisciplinary approach including osteological, geochemical and biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from control skeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae with detection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, the preservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP) and multiple locus variable number tandem repeat analysis (MLVA). Three of the five cases proved to be infected with SNP type 3I-1, ancestral to contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From the remaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotope analysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all three individuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as the precursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains. Thus we show that type 2F had also spread westwards to Britain by the early medieval period.     

Development of VNTR markers for two Fusarium graminearum clade species

Using a bioinformatics approach, we developed 10 variable number of tandem repeat (VNTR) markers for Fusarium graminearum and Fusarium asiaticum useful for population genetic studies. Repeat sequences in the genome sequence of F. graminearum were identified by a tandem repeat finding program. Length polymorphisms at 54 loci were examined for five strains each from the United States, Italy and China. From these 54 loci, 10 were selected based on polymorphisms detected across species, ease of scoring, and their dispersed location in the genome.     

Insights to Genetic Characterization Tools for Epidemiological Tracking of Francisella tularensis in Sweden

Tularaemia, caused by the bacterium Francisella tularensis, is endemic in Sweden and is poorly understood. The aim of this study was to evaluate the effectiveness of three different genetic typing systems to link a genetic type to the source and place of tularemia infection in Sweden. Canonical single nucleotide polymorphisms (canSNPs), MLVA including five variable number of tandem repeat loci and PmeI-PFGE were tested on 127 F. tularensis positive specimens collected from Swedish case-patients. All three typing methods identified two major genetic groups with near-perfect agreement. Higher genetic resolution was obtained with canSNP and MLVA compared to PFGE; F. tularensis samples were first assigned into ten phylogroups based on canSNPs followed by 33 unique MLVA types. Phylogroups were geographically analysed to reveal complex phylogeographic patterns in Sweden. The extensive phylogenetic diversity found within individual counties posed a challenge to linking specific genetic types with specific geographic locations. Despite this, a single phylogroup (B.22), defined by a SNP marker specific to a lone Swedish sequenced strain, did link genetic type with a likely geographic place. This result suggests that SNP markers, highly specific to a particular reference genome, may be found most frequently among samples recovered from the same location where the reference genome originated. This insight compels us to consider whole-genome sequencing (WGS) as the appropriate tool for effectively linking specific genetic type to geography. Comparing the WGS of an unknown sample to WGS databases of archived Swedish strains maximizes the likelihood of revealing those rare geographically informative SNPs.     

Impairment of alternate pathway (CD2) of T cell activation in leprosy

Recent studies in basic immunology have been directed towards the understanding of the mechanism of T cell activation. T cells can be activated to proliferatevia the classical pathway through the antigen receptor (CD3-Ti) orvia the alternate pathway through the CD2 receptor. Since immunologic unresponsiveness in lepromatous leprosy is considered to be due to the inability of T cells to proliferate upon stimulation, we have been interested in the nature of these receptors and the activation pathways in lymphocytes of leprosy patients. In the present investigation we demonstrate: (i) CD2 receptor (E-receptor) is downregulated in bacterial index positive lepromatous leprosy patients. (ii) The alternate pathway of T cell activation is impaired in lepromatous patients as revealed by the inability of their lymphocytes to proliferate in response to a pair of mitogenic anti-CD2 monoclonals. (iii) The addition of recombinant interleukin 2in vitro restores the ability of lymphocytes from lepromatous patients to proliferate in response to anti-CD2 antibodies. (iv) Interestingly, CD2 modulation and the associated functional impairment could be brought about in peripheral blood lymphocytes from normal subjects by prior treatment withMycobacterium leprae in vitro. This approach would be useful in understanding the molecular events leading to the defective T cell functions in leprosy.     

Tracing isolates of bacterial species by multilocus variable number of tandem repeat analysis (MLVA)

All bacterial genomes contain multiple loci of repetitive DNA. Repeat unit sizes and repeat sequences may vary when multiple loci are considered for different isolates of an individual microbial species. Moreover, it has been documented on many occasions that the number of repeat units per locus is a strain-defining parameter. Consequently, there is isolate-specificity in the number of repeats per locus when different strains of a given bacterial species are compared. The experimental assessment of this variability for a number of different loci has been called 'multilocus variable number of tandem repeat analysis' (MLVA). The approach can be supported or extended by locus-specific DNA sequencing for establishing mutations in the individual repeat units, which usually enhances the resolution of the approach considerably. Essentially, MLVA with or without supportive sequencing has been developed for all of the medically relevant bacterial species and can be used effectively for tracing outbreaks or other forms of bacterial dissemination. MLVA is a modern, timely and versatile bacterial typing methodology.     

Rapid and efficient resolution of parentage by amplification of short tandem repeats.

Short tandem repeat (STR) loci are highly informative polymorphic loci that are gaining popularity for identity testing. We have conducted parentage testing by using nine STR loci on 50 paternity trios that had been previously tested using VNTR loci. These nine unlinked STR loci are amplified in three multiplex reactions and, when examined for genetic informativeness, provide a combined average power of exclusion of 99.73% (Caucasian data). The informative value of the selected loci is based on extensive STR typing of four racial/ethnic populations. In 37 of the 50 cases, paternity could not be excluded by any of the loci. In the remaining 13 cases, paternity was excluded by at least two of the STR markers. The probability of paternity calculated for the alleged father of each matching trio was > 99% in 36 of the 37 inclusion cases. All data agreed with the results reported using VNTR loci and conventional Southern technology. Our studies validate the use of DNA typing with STR loci for parentage testing, thus providing an accurate, highly sensitive, and rapid assay.     

Genetic diversity of two African and sixteen South American populations determined on the basis of six hypervariable loci

A total of 582 individuals (1,164 chromosomes) from two African, eight African-derived South American, five South American Amerindian, and three Brazilian urban populations were studied at four variable number of tandem repeat (VNTR) and two short tandem repeat (STR) hypervariable loci. These two sets of loci did not show distinct allele profiles, which might be expected if different processes promoted their molecular differentiation. The two African groups showed little difference between them, and their intrapopulational variation was similar to those obtained in the African-derived South American communities. The latter showed different degrees of interpopulation variability, despite the fact that they presented almost identical average degrees of non-African admixture. The F_ST single locus estimates differed in the five sets of populations, probably due to genetic drift, indicating the need to consider population structure in the evaluation of their total variability. A high interpopulational diversity was found among Amerindian populations in relation to Brazilian African-derived isolated communities. This is probably a consequence of the differences in the patterns of gene flow and genetic drift that each of these semi-isolated groups experienced. Am J Phys Anthropol 109:425–437, 1999. © 1999 Wiley-Liss, Inc.     

Multiple-locus variable-number tandem repeat analysis for strain typing of Clostridium perfringens

Clostridium perfringens is ubiquitous in the environment and causes diseases in man and animals, with syndromes ranging from enteritis, enterotoxemia, and sudden death to food poisoning and gas gangrene. Understanding the epidemiology of these infections and of the evolution of virulence in C. perfringens necessitate an efficient, time and cost effective strain typing method. Multiple-locus variable-number tandem repeat analysis (MLVA) has been applied to typing of other pathogens and we describe here the development of a MLVA scheme for C. perfringens. We characterized five variable tandem repeat (VNTR) loci, four of which are contained within protein encoding genes and screened 112 C. perfringens isolates to evaluate typability, reproducibility, and discriminatory power of the scheme. All the isolates were assigned a MLVA genotype and the technique has excellent reproducibility, with a numerical index of discrimination for the five VNTR loci of 0.995. Thus MLVA is an efficient tool for C. perfringens strain typing, and being PCR based makes it rapid, easy, and cost effective. In addition, it can be employed in epidemiological, ecological, and evolutionary investigations of the organism.     

Phylogenetic relationship of the populations within and around Japan using 105 short tandem repeat polymorphic loci

We have analyzed 105 autosomal polymorphic short tandem repeat (STR) loci for nine East and South-eastern Asian populations (two Japanese, five Han Chinese, Thai, and Burmese populations) and a Caucasian population using a multiplex PCR typing system. All the STR loci are genomewide tetranucleotide repeat markers of which the total number of observed alleles and the observed heterozygosity were 756 and 0.743, respectively, for Japanese populations. Phylogenetic analysis for these allele frequency data suggested that the Japanese populations are more closely related with southern Chinese populations than central and/or northern ones. STRUCTURE program analysis revealed the almost clearly divided and accountable population structure at K=2–6, that the two Japanese populations always formed one group separated from the other populations and never belong to different groups at K≥3. Furthermore, our new allele frequency data for 91 loci were analyzed with those for 52 worldwide populations published by previous studies. Phylogenetic and multidimensional scaling (MDS) analyses indicated that Asian populations with large population size (six Han Chinese, three Japanese, two Southeast Asia) formed one distinct cluster and are closer to each other than other ethnic minorities in east and Southeast Asia. This pattern may be the caviar of comparing populations with greatly differing population sizes when STR loci were analyzed.     

Variants of the anti-Müllerian hormone gene in a compound heterozygote with the persistent Müllerian duct syndrome and his family

Summary The human genome contains a large number of interspersed simple repeat sequences that are variable in length and can therefore serve as highly informative, polymorphic markers. Typing procedures include conventional multilocus and single locus probing, and polymerase chain reaction aided analysis. We have identified simple sequences in a cosmid clone stemming from the human Y chromosome and consisting of (gata)_n repeats. We have compared these with two equivalent simple repeat loci from chromosome 12. After amplifying the tandemly repeated motifs, we detected between four and eight different alleles at each of the three loci. Codominant inheritance of the alleles was established in family studies and the informativity of the simple repeat loci was determined by typing unrelated individuals. The polymorphisms are suitable for application in linkage studies, practical forensic case work, deficiency cases in paternity determination, and for studying ethnological questions. The mutational mechanisms that bring about changes in simple repeats located both on the autosomes and on the sex chromosomes, are discussed.Professor Dr. Otto Prokop (Humboldt-Universität Berlin) on the occasion of his 70th birthday     

Genotypic characterization to identify markers associated with putative hypervirulence in Swedish Escherichia coli O157:H7 cattle strains

Aims: To establish whether investigated subtyping methods could identify any specific characteristics that distinguish Swedish VTEC O157:H7 strains isolated from cattle farms associated with human enterohaemorrhagic Escherichia coli (EHEC) cases from cattle strains isolated in prevalence studies. Methods and Results: Strains (n = 32) isolated in a dairy herd prevalence study and strains isolated from farms associated with human cases (n = 13) were subjected to typing. Partial sequencing of the vtx₂ genes could not identify any unique variants of vtx₂ or vtx_2c in strains associated with human cases. A specific variant of VTEC O157:H7, which was overrepresented among farms associated with human cases (P = 0·01), was by two different single‐nucleotide‐polymorphism (SNP) assays identified as clade 8, a subgroup of VTEC O157:H7 strains considered to be putatively hypervirulent. Multi‐locus variable number tandem repeat analysis (MLVA) typing of all strains produced similar results as pulsed‐field gel electrophoresis (PFGE) typing regarding clustering of the strains, but MLVA distinguished slightly better among strains than PFGE. Conclusion: In Sweden, VTEC O157:H7 strains from the putatively hypervirulent clade 8 are overrepresented among isolates from cattle farms associated with human cases compared with VTEC O157:H7 strains isolated in prevalence studies. Significance and Impact of the Study: Real‐time PCR SNP typing for clade 8 can be used to identify cattle farms that are at higher risk of causing EHEC infections in humans.     

O-diphenoloxidase concentrations in leprosy.

O-diphenoloxidase activity was studied in 15 patients with lepromatous leprosy, 15 with tuberculois leprosy, and 15 controls. O-diphenoloxidase isolated from skin and serum samples of patients with lepromatous leprosy had the specificity of a bacterially derived enzyme and not that of a mammalian-derived enzyme. Only the patients who had lepromatous leprosy for over two years showed enzyme activity in serum, though all showed it in skin tissue. O-diphenoloxidase activity in serum may be a useful diagnostic marker of lepromatous leprosy.     

Analysis of mosquito bloodmeals by DNA profiling

Abstract. Human specific genetic markers have been used to profile the human DNA found within a mosquito bloodmeal. In this technique, variable numbers of tandem repeat (VNTR) sequences are employed to prime amplification of human DNA in the polymerase chain reaction (PCR) and the radiolabeled products are analysed by high resolution denaturing gel electrophoresis. Matching of DNA profiles allows identification of the individual human host. Bloodmeals of 125 female Anopheles gambiae Giles mosquitoes, caught dead or alive in verandah-trap huts wherein two people had slept overnight protected by intact insecticide-impregnated bednets, were analysed: thirty-five out of thirty-nine profiles generated were identical to those of the sleepers under the nets. Thus the blood-fed mosquitoes found after impregnated nets have been used cannot, in most cases, be explained away by entry of already fed mosquitoes into the huts.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

Nine polymorphic STR loci in the HLA region in the Shaanxi Han population of China

A large number of microsatellite genetic markers have been identified in the human leukocyte antigen (HLA) region. We investigated genetic polymorphism of the nine short tandem repeat (STR) loci (D6S276, MOGCA, D6S265, MIB, D6S273, G51152, TAP1CA, RING3CA, and D6S291) in the HLA region in the Shaanxi Han population. Using a fluorescence-labeled multiplex-PCR STR typing method, 6-13 alleles were detected in these nine STR loci in 150 unrelated Han Chinese from the region of Shaanxi, China. The distributions of the genotypes at these nine loci were in Hardy-Weinberg equilibrium. We conclude that these nine STR loci have a high level of genetic polymorphism; they would be useful for population genetic studies, pre-transplantation HLA typing, forensic and paternity testing, etc.