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Results for “Multilocus sequence typing”

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PubMed2004

Population structure of Salmonella investigated by amplified fragment length polymorphism.

AIMS: This study was undertaken to investigate the usefulness of amplified fragment length polymorphism (AFLP) in determining the population structure of Salmonella. METHODS AND RESULTS: A total of 89 strains were subjected to AFLP analysis using the enzymes BglII and BspDI, a combination that is novel in Salmonella. Both species S. bongori and S. enterica and all subsp. of S. enterica were represented with emphasis on S. enterica subsp. enterica using a local strain collection and strains from the Salmonella Reference Collection B (SARB). The amplified fragments were used in a band-based cluster analysis. The tree resulting from the subgroup analysis clearly separated all subgroups with high bootstrap values with the species S. bongori being the most distantly related of the subgroups. The tree resulting from the analysis of the SARB collection showed that some serotypes are very clonal whereas others are highly divergent. CONCLUSIONS: AFLP clearly clustered strains representing the subgroups of Salmonella together with high bootstrap values and the serotypes of subspecies enterica were divided into polyphyletic or monophyletic types corresponding well with multilocus enzyme electrophoresis (MLEE) and sequence-based studies of the population structure in Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP with the enzyme combination BglII and BspDI allows discrimination of individual strains and provides evidence for the usefulness of AFLP in studies of population structure in Salmonella.

Base Sequence↗

PubMed2025

Understanding recurrence in Mycobacterium avium complex pulmonary disease: genotypic strategies to support clinical decision-making.

Pulmonary disease caused by Mycobacterium avium complex (MAC-PD) is a chronic, recurrent disease, and its high recurrence rate after treatment makes clinical management difficult. Distinguishing whether recurrence is due to persistence of existing strains or reinfection with new strains is essential for establishing treatment strategies, preventing overuse of antimicrobials, and establishing infection control measures. According to reports, 54%-74% of MAC-PD recurrence is due to reinfection, which may be mainly related to environmental reservoirs such as household water supply. In this review, we present various clinical scenarios in which MAC-PD recurrence may occur and examine genotyping techniques as a strategy to distinguish and respond to them. From traditional methods such as IS1245-based restriction fragment length polymorphism, pulsed-field gel electrophoresis, and hsp65 and rpoB gene sequencing to high-resolution analysis techniques such as multilocus sequence testing and whole-genome sequencing, the latest molecular typing methods are comprehensively summarized. Integrating these genotype data into clinical settings, standardizing single-nucleotide polymorphism-based interpretation thresholds, and promoting the establishment of a global MAC strain database will make a substantial contribution to more accurately distinguishing the recurrence mechanisms of MAC-PD and establishing personalized treatment strategies.IMPORTANCEThe global burden of nontuberculous mycobacterial pulmonary disease (PD) is increasing, with Mycobacterium avium (MAC)-PD being the most prevalent and clinically challenging form. Its low treatment success rates, high frequency of recurrence, and persistent environmental exposure complicate both diagnosis and management. A critical clinical issue is determining whether recurrence represents true relapse, due to persistence of the original strain, or reinfection with a new strain, as this guides treatment and prevents overtreatment. Genotypic strategies capable of resolving strain-level differences can improve diagnostic accuracy, prevent misclassification, and ultimately support more informed treatment decisions. Therefore, integrating genotyping data into clinical workflows, standardizing single-nucleotide polymorphism thresholds, and establishing a global MAC strain database will not only support personalized treatment but also enhance the broader public health response to this disease.

Humans↗

PubMed2004

Historical and contemporary multilocus population structure of Ascochyta rabiei (teleomorph: Didymella rabiei) in the Pacific Northwest of the United States.

The historical and contemporary population genetic structure of the chickpea Ascochyta blight pathogen, Ascochyta rabiei (teleomorph: Didymella rabiei), was determined in the US Pacific Northwest (PNW) using 17 putative AFLP loci, four genetically characterized, sequence-tagged microsatellite loci (STMS) and the mating type locus (MAT). A single multilocus genotype of A. rabiei (MAT1-1) was detected in 1983, which represented the first recorded appearance of Ascochyta blight of chickpea in the PNW. During the following year many additional alleles, including the other mating type allele (MAT1-2), were detected. By 1987, all alleles currently found in the PNW had been introduced. Highly significant genetic differentiation was detected among contemporary subpopulations from different hosts and geographical locations indicating restricted gene flow and/or genetic drift occurring within and among subpopulations and possible selection by host cultivar. Two distinct populations were inferred with high posterior probability which correlated to host of origin and date of sample using Bayesian model-based population structure analyses of multilocus genotypes. Allele frequencies, genotype distributions and population assignment probabilities were significantly different between the historical and contemporary samples of isolates and between isolates sampled from a resistance screening nursery and those sampled from commercial chickpea fields. A random mating model could not be rejected in any subpopulation, indicating the importance of the sexual stage of the fungus both as a source of primary inoculum for Ascochyta blight epidemics and potentially adaptive genotypic diversity.

Ascomycota↗

PubMed2004

Molecular typing of Yersinia frederiksenii strains by means of 16s rDNA and gyrB genes sequence analyses.

The phenospecies Yersinia frederiksenii is known to comprise three genospecies, indistinguishable on the basis of phenotypic characteristics. In previous works, 13 strains, identified biochemically as Y. frederiksenii, were characterized using Multilocus enzyme electrophoresis and Ribotyping. In order to elucidate the phylogenetic position of these strains we performed their molecular typing by means of 16S rDNA and gyrB sequences analyses. Results demonstrated that gyrB is a better phylogenetic marker than 16S rDNA. The classification achieved by gyrB sequences analyses was in agreement with results obtained with more laborious methods. Moreover, a good phylogenetic identification could be reached also with partial gyrB sequences of only 350 bp.

Bacterial Typing Techniques↗

PubMed1999

A multilocus genotyping assay for candidate markers of cardiovascular disease risk.

A number of chronic diseases, including cardiovascular disease, appear to have a multifactorial genetic risk component. Consequently, techniques are needed to facilitate evaluation of complex genetic risk factors in large cohorts. We have designed a prototype assay for genotyping a panel of 35 biallelic sites that represent variation within 15 genes from biochemical pathways implicated in the development and progression of cardiovascular disease. Each DNA sample is amplified using two multiplex polymerase chain reactions, and the alleles are genotyped simultaneously using an array of immobilized, sequence-specific oligonucleotide probes. This multilocus assay was applied to two types of cohorts. Population frequencies for the markers were estimated using 496 unrelated individuals from a family-based cohort, and the observed values were consistent with previous reports. Linkage disequilibrium between consecutive pairs of markers within the apoCIII, LPL, and ELAM genes was also estimated. A preliminary analysis of single and pairwise locus associations with severity of atherosclerosis was performed using a composite cohort of 142 individuals for whom quantitative angiography data were available; evaluation of the potentially interesting associations observed will require analysis of an independent and larger cohort. This assay format provides a research tool for studies of multilocus genetic risk factors in large cardiovascular disease cohorts, and for the subsequent development of diagnostic tests.

Adult↗

PubMed2001

Francisella tularensis strain typing using multiple-locus, variable-number tandem repeat analysis.

Francisella tularensis, the etiological agent of tularemia, is found throughout the Northern hemisphere. After analyzing the F. tularensis genomic sequence for potential variable-number tandem repeats (VNTRs), we developed a multilocus VNTR analysis (MLVA) typing system for this pathogen. Variation was detected at six VNTR loci in a set of 56 isolates from California, Oklahoma, Arizona, and Oregon and the F. tularensis live vaccine strain. PCR assays revealed diversity at these loci with total allele numbers ranging from 2 to 20, and Nei's diversity index values ranging from 0.36 to 0.93. Cluster analysis identified two genetically distinct groups consistent with the current biovar classification system of F. tularensis. These findings suggest that these VNTR markers are useful for identifying F. tularensis isolates at this taxonomic level. In this study, biovar B isolates were less diverse than those in biovar A, possibly reflecting the history of tularemia in North America. Seven isolates from a recent epizootic in Maricopa County, Ariz., were identical at all VNTR marker loci. Their identity, even at a hypervariable VNTR locus, indicates a common source of infection. This demonstrates the applicability of MLVA for rapid characterization and identification of outbreak isolates. Future construction of reference databases will allow faster outbreak tracking as well as providing a foundation for deciphering global genetic relationships.

Animals↗

PubMed1997

Molecular population genetic analysis of a Streptococcus pyogenes bacteriophage-encoded hyaluronidase gene: recombination contributes to allelic variation.

Many strains of the human pathogenic bacterium Streptococcus pyogenes produce hyaluronidase, an enzyme that degrades hyaluronic acid, a major component of the extracellular matrix. Degradation of hyaluronic acid is thought to aid in host tissue invasion and dissemination of S. pyogenes. The molecular population genetics of the bacteriophage-encoded hyaluronidase gene (hyl) was analysed by sequencing the gene from 13 streptococcal strains representing seven well-differentiated multilocus enzyme electrophoretic types and eight M or T protein serotypes. Substantial levels of allelic polymorphism were identified, and the analysis found strong statistical evidence that recombinational processes have contributed to the generation of molecular variation in this gene. A 111 base pair segment of hyl encoding a collagenous motif, that may bind collagen, was absent in a serotype M14 isolate and 13 serotype M18 multilocus enzyme electrophoretic type 20 strains examined. The analysis provides a molecular population genetics framework for studies examining the role of naturally occurring hyaluronidase variation in host-pathogen interactions.

Alleles↗

PubMed2001

Sequence type analysis and recombinational tests (START).

UNLABELLED: The 32-bit Windows application START is implemented using Visual Basic and C(++) and performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. These analyses include data summary, lineage assignment, and tests for recombination and selection. AVAILABILITY: START is available at http://outbreak.ceid.ox.ac.uk/software.htm. CONTACT: keith.jolley@ceid.ox.ac.uk

Databases, Nucleic Acid↗

PubMed2002

Microsatellite analysis of Toxoplasma gondii shows considerable polymorphism structured into two main clonal groups.

Previous studies on Toxoplasma gondii population structure, based essentially on multilocus restriction fragment length polymorphism analysis or on multilocus enzyme electrophoresis, indicated that T. gondii comprises three clonal lineages. These studies showed a weak polymorphism of the markers (2-4 alleles by locus). In this study, we used eight microsatellite markers to type 84 independent isolates from humans and animals. Two microsatellite markers were present in the introns of two genes, one coding for beta-tubulin and the other for myosin A, and six were found in expressed sequence tags. With 3-16 alleles detected, these markers can be considered as the most discriminating multilocus single-copy markers available for typing T. gondii isolates. This high discriminatory power of microsatellites made it possible to detect mixed infections and epidemiologically related isolates. Evolutionary genetic analyses of diversity show that the T. gondii population structure consists of only two clonal lineages that can be equated to discrete typing units, but there is some evidence of occasional genetic exchange that could explain why one of these discrete typing units is less clearly individualised than the other.

Animals↗

PubMed1995

State of the art: typing Mycobacterium tuberculosis.

After years of decline, infections due to Mycobacterium tuberculosis appear to be increasing in the developed world. This has led to a search for improved methods of typing strains in order to answer new questions concerning the epidemiology of tuberculosis. Owing to the phenotypic and genotypic homogeneity of the species, classical fingerprinting methods, including phage typing, serotyping and multilocus enzyme electrophoresis, have proven ineffective. However, new methods based on the analysis of repetitive DNA sequences have been introduced and are now being used to elucidate the emerging pattern of disease.

Bacterial Typing Techniques↗

PubMed1995

Extreme clonal uniformity of Phoxinus eos/neogaeus gynogens (pisces: Cyprinidae) among variable habitats in northern Minnesota beaver ponds.

Genetic surveys of parthenogenetic vertebrate populations have demonstrated a common pattern of relatively high degrees of clonal variation and the coexistence of numerous clones. In striking contrast, the Phoxinus eos/Phoxinus neogaeus/hybrid gynogen complex of cyprinid fishes exhibits no clonal variation within a northern Minnesota drainage characterized by successional beaver ponds. Gynogens were sampled from three habitats in each of four different pond types in a single drainage in Voyageurs National Park, Minnesota. The abundance of gynogens relative to sexual dace varied with pond type, being least common in deep upland ponds and most common in shallow, collapsed, lowland ponds (13.4% and 48.6%, respectively). Simple-sequence multilocus DNA fingerprinting of 464 individual gynogens detected one, and only one, clone. DNA fingerprints, generated sequentially by using three oligonucleotide probes, (CAC)5, (GACA)4, and the Jeffreys' 33.15 probe, all revealed the same unprecedented lack of variation. The extreme lack of clonal diversity in these gynogens across a range of habitat types does not fit the general pattern of high clonal diversity found within populations of other vertebrate parthenogens.

Animals↗

PubMed2002

Diversity of rhizobia from nodules of the leguminous tree Gliricidia sepium, a natural host of Rhizobium tropici.

The Rhizobium species that nodulate the legume tree Gliricidia sepium were analyzed by phenotypic characteristics (including nodule formation in different hosts), PCR-RFLP patterns and sequences of 16S rRNA genes, multilocus enzyme electrophoresis, and plasmid patterns. Strains of Rhizobium tropici type A and B, Sinorhizobium spp., and Rhizobium etli bv. phaseoli were encountered in G. sepium nodules and their presence depended on the site sampled.

Fabaceae↗

PubMed1994

Non-congruent relationships between variation in emm gene sequences and the population genetic structure of group A streptococci.

To examine the molecular population genetics of the M protein family of Streptococcus pyogenes (group A Streptococcus), the 5' regions of polymerase chain reaction-amplified emm products from 79 M serotypes were sequenced and the phylogeny was compared to estimates of overall genetic relationships among strains determined by multilocus enzyme electrophoresis. Although the 5' emm sequences from several strains designated as distinct M types were identical or almost identical, the overall pattern is characterized by very extensive variation. The composition of distinct emm sequence clusters generally parallels the ability of strains to express serum opacity factor and in some cases historical associations of certain M types with acute rheumatic fever, but not with M types classified as nephritogenic. For many strains there is a lack of congruency between variation in 5' emm sequences and estimates of overall chromosomal relationships, which is undoubtedly due to horizontal transfer and recombination of emm sequences. The results of these studies provide insights into the nature and extent of emm sequence variation and describe how this variation 'maps' onto the population genetic structure of extant S. pyogenes lineages. The complexity of emm sequence and streptococcal cell lineage relationships revealed by this analysis has significant implications for understanding evolutionary events generating strain diversity and the epidemiology of S. pyogenes diseases.

Amino Acid Sequence↗

PubMed1995

Listeria monocytogenes exists in at least three evolutionary lines: evidence from flagellin, invasive associated protein and listeriolysin O genes.

Regions of the genes encoding flagellin (flaA), the invasive associated protein (iap), listeriolysin O (hly) and 23S rRNA were sequenced for a range of Listeria monocytogenes isolates of different origin and serotypes. Several nucleotide sequence variations were found in the flaA, iap and hly genes. No differences were found for the rRNA genes, but our approach does not exclude the existence of differences between single copies of these genes. Based on the sequence differences, the L. monocytogenes strains can be divided into three distinct sequence types. Further, the presence of only a small number of sequence differences within each group indicates a strong degree of conservation within the groups. There was a complete correspondence among the groups of strains formed according to the analysis of the flaA, iap and hly genes, and the grouping correlates with serotype, pulsed field gel electrophoretic and multilocus enzyme electrophoretic data. Analysis of the region encoding the threonine-asparagine repeat units in the iap gene revealed some striking features. Sequence type 1 strains were found to have 16-17 repeats, sequence type 2 strains had 16-20 repeats whereas the two sequence type 3 strains analysed had only 11 repeats. Furthermore, within a 19 bp segment there was a 37% difference between the sequences of type 1 and 2 strains and that segment was absent in type 3 strains. Within the threonine-asparagine repeat region the nucleotide differences gave rise to four amino acid changes; however, all were changes among the three amino acids present in the repeat structure indicating a strong selective pressure on the composition of this region.

Animals↗

PubMed1998

Hypervariability generated by natural selection in an extracellular complement-inhibiting protein of serotype M1 strains of group A Streptococcus.

In many countries, M1 strains of the human pathogenic bacterium group A Streptococcus are the most common serotype recovered from patients with invasive disease episodes. Strains of this serotype express an extracellular protein that inhibits complement [streptococcal inhibitor of complement (Sic)] and is therefore believed to be a virulence factor. Comparative sequence analysis of the 915-bp sic gene in 165 M1 organisms recovered from diverse localities and infection types identified 62 alleles. Inasmuch as multilocus enzyme electrophoresis and pulsed-field gel electrophoresis previously showed that most M1 organisms represent a distinct streptococcal clone, the extent of sic gene polymorphism was unexpected. The level of polymorphism greatly exceeds that recorded for all other genes examined in serotype M1 strains. All insertions and deletions are in frame, and virtually all nucleotide substitutions alter the amino acid sequence of the Sic protein. These molecular features indicate that structural change in Sic is mediated by natural selection. Study of 70 strains recovered from two temporally distinct epidemics of streptococcal infections in the former East Germany found little sharing of Sic variants among strains recovered in the different time periods. Taken together, the data indicate that sic is a uniquely variable gene and provide insight into a potential molecular mechanism contributing to fluctuations in streptococcal disease frequency and severity.

Antigens, Bacterial↗

PubMed2005

Stability of PorA during a meningococcal disease epidemic.

Meningococci causing New Zealand's epidemic, which began in 1991, are defined as group B, serosubtype P1.4 (subtype P1.7-2,4), belonging to the ST-41/ST-44 complex, lineage III. Of the 2,358 group B isolates obtained from disease cases from 1991 through 2003, 85.7% (2,021 of 2,358) were determined to be serosubtype P1.4. Of the remaining isolates, 156 (6.6%) were not serosubtypeable (NST). Molecular analysis of the porA gene from these B:NST meningococcal isolates was used to determine the reason. Most NST isolates (156, 88.5%) expressed a PorA that was distinct from P1.7-2,4 PorA. Fifteen isolates expressed variants of P1.7-2,4 PorA, and a further three expressed P1.7-2,4 PorA without any sequence variation. These three isolates expressed P1.7-2,4 PorA at very low levels, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and showed variation in the porA promoter region. Among the 15 meningococcal isolates expressing variants of P1.7-2,4 PorA, 11 different sequence variations were found. Compared with the P1.7-2,4 PorA sequence, the sequences of these variants contained deletions, insertions, or single-nucleotide substitutions in the VR2 region of the protein. Multilocus restriction typing was used to assess the clonal derivations of B:NST case isolates. Meningococcal isolates expressing distinct PorA proteins belonged mostly to clonal types that were unrelated to the epidemic strain, whereas all meningococcal isolates expressing variants of P1.7-2,4 PorA belonged to the ST-41/ST-44 complex, lineage III. These results, together with those obtained serologically, demonstrate that the P1.7-2,4 PorA protein of meningococci responsible for New Zealand's epidemic has remained relatively stable over 13 years and support the use of a strain-specific outer membrane vesicle vaccine to control the epidemic.

Amino Acid Sequence↗

PubMed1996

Rapid determination of outbreak-related strains of Neisseria meningitidis by repetitive element-based polymerase chain reaction genotyping.

Outbreaks of invasive disease caused by Neisseria meningitidis have increased in the United States in the 1990s. Repetitive element-based polymerase chain reaction (rep-PCR), a recently developed genotyping method, was used to evaluate a group of 8 outbreak-related and 35 other meningococcal isolates previously typed by multilocus enzyme electrophoresis (MLEE). All were serogroup B or C. Sets of genotypes were generated using primers based on either of two different repetitive sequences. Genotype sets were analyzed in a blinded fashion. Each set correctly identified outbreak-related isolates. Among the other 35 isolates, rep-PCR delineated 14 and 13 strains, respectively, in the two sets of genotypes. Seventeen electrophoretic types had been delineated by MLEE. Rep-PCR holds promise as a rapid, genome-based typing method for delineation of apparent outbreaks of meningococcal disease.

Disease Outbreaks↗

PubMed2005

Multiplex PCR for typing strains of Toxoplasma gondii.

A multiplex PCR assay was designed for multilocus strain typing of Toxoplasma gondii based on length polymorphism of five microsatellite markers. Eight T. gondii strains already sequenced at these five markers were used as control isolates. This method is simple, rapid, reproducible, and adapted to a large set of isolates.

Animals↗