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Results for “Multilocus sequence typing”

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PubMed1996

Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons.

Multilocus enzyme electrophoresis (MEE) analysis and comparisons of nearly complete 16S rRNA gene sequences (1416 nucleotide positions) were used to evaluate phylogenetic relationships among Serpulina hyodysenteriae strain B78T, S. innocens strain B256T, Brachyspira aalborgi strain 513AT, and eight uncharacterised strains of swine, avian, and human intestinal spirochaetes. From MEE analysis, nine strains could be assigned to five groups containing other intestinal spirochaetes (genetic distances between groups = 0.6-0.9). Chicken spirochaete strain C1 and B. aalborgi 513AT represented unique electrophoretic types and formed their own MEE groups. Despite MEE differences, the 11 strains had highly similar (96.3-99.9%) 16S rRNA sequences. These findings point out limitations of both MEE analysis and 16S rRNA sequence comparisons when used as solitary techniques for classifying intestinal spirochaetes related to Brachyspira/Serpulina species.

Alleles↗

PubMed2001

Allelic diversity and recombination in Campylobacter jejuni.

The allelic diversity and population structure of Campylobacter jejuni were studied by multilocus nucleotide sequence analysis. Sequences from seven housekeeping genes were obtained from 32 C. jejuni isolates isolated from enteritis patients in Germany, Hungary, Thailand, and the United States. Also included was strain NCTC 11168, the complete genomic sequence of which has recently been published. For all loci analyzed, multiple strains carried identical alleles. The frequency of synonymous and nonsynonymous sequence polymorphisms was low. The number of unique alleles per locus ranged from 9 to 15. These alleles occurred in 31 different combinations (sequence types), so that all but two pairs of strains could be distinguished from each other. Sequences were analyzed for evidence of recombination by the homoplasy test and split decomposition. These analyses showed that intraspecific recombination is frequent in C. jejuni and has generated extensive diversity of allelic profiles from a small number of polymorphic nucleotides.

Alleles↗

PubMed1997

Ubiquitous distribution of the competence related genes comA and comC among isolates of Streptococcus pneumoniae.

DNA probes specific for the pneumococcal competence regulatory genes comA and comC were used to test the presence of these genes in 214 pneumococcal isolates selected to represent a wide variety of clonal types and genetic backgrounds (as defined by chromosomal macrorestriction patterns and multilocus enzyme analysis), a variety of serotypes, isolation dates (between 1916 and 1996), geographic origins (on four continents), as well as different clinical origins (including both infection sites and colonization sites). Each isolate gave positive signal with both DNA probes. The comA and comC genes were never on the same SmaI restriction fragment and the comA gene showed a considerable degree of polymorphism from one strain to another. While DNA sequencing of 50 of the isolates have identified three distinct alleles of the comC gene, the number of mutations within the leader peptide were minimal. The results suggest that the mechanism to undergo genetic transformation is widespread within the species of Streptococcus pneumoniae.

Alleles↗

PubMed2000

Phylogeny of Porphyromonas gingivalis by ribosomal intergenic spacer region analysis.

Periodontitis has been associated with the presence of Porphyromonas gingivalis, and previous studies have shown phenotypic differences in the pathogenicities of strains of P. gingivalis. An accurate and comprehensive phylogeny of strains of P. gingivalis would be useful in determining if there is an evolutionary basis to pathogenicity in this species. Previous phylogenies of P. gingivalis strains based on random amplified polymorphic DNA (RAPD) analysis and multilocus enzyme electrophoresis (MLEE) show little agreement. While the 16S ribosomal gene is the standard for phylogenetic reconstruction among bacterial species, it is insufficiently variable for this purpose. In the present study, the phylogeny of P. gingivalis was constructed on the basis of the sequence of the most variable region of the ribosomal operon, the intergenic spacer region (ISR). Heteroduplex analysis of the ISR has been used to study the variability of P. gingivalis strains in periodontitis. In the present study, typing by heteroduplex analysis was compared to ISR sequence-based phylogeny and close agreement was observed. The two strains of P. gingivalis whose heteroduplex types are strongly associated with periodontitis were found to be closely related and were well separated from strains whose heteroduplex types are less strongly associated with disease, suggesting a relationship between pathogenicity and phylogeny.

Electrophoresis↗

PubMed1994

Isolation and characterization of hypervariable sequences from tsetse fly genome.

A Glossina brevipalpis Newstead genomic library, constructed using a Charomid 9-36 vector, was used to isolate putative clones that hybridize to polymorphic regions of the tsetse genome. Five types of probes, that reveal individual DNA polymorphic in humans and higher animal species, were used to screen 300 tsetse Charomid clones; 15% of the clones hybridized with at least one probe. Twenty four recombinants were further characterized by Southern blotting hybridization using DNA isolated from individual tsetse fly from Glossina morsitans centralis Machado. Two classes of DNA profiles were obtained upon hybridization with the recombinant inserts. The first, termed the "multilocus profile" displayed a complex DNA pattern of multiple components whilst the second, referred to as the "single locus profile" revealed one or two bands in each individual. Of the 24 recombinant inserts tested, 13 were multilocus probes and the remainder were single locus probes, each of which hybridized to a single location when G. m. centralis DNA had been cleaved with EcoRI. These single locus probes revealed a low level of genetic variability among individual flies from an inbred colony. The hybridization profiles using multilocus and single locus probes were also obtained on DNA from individual Glossina palpalis palpalis Robineau-Desvoidy and Glossina palpalis gambienis Vanderplank and some of the G. brevipalpis recombinant clones also detected multilocus profiles in honey bees and man.

Animals↗

PubMed2000

Molecular comparison of extraintestinal Escherichia coli isolates of the same electrophoretic lineages from humans and domestic animals.

Molecular typing methods were used to characterize 38 Escherichia coli strains that originally were isolated from extraintestinal infections and represented 5 multilocus enzyme electrophoretic types (ETs) recovered from both humans and animals. Within each ET, the human and animal isolates did not consistently segregate by host group, according to individual virulence factors (VFs), composite VF-serotype profiles, or pulsed-field gel electrophoresis profiles. Several close matches with respect to VF-serotype profiles were identified between human and canine isolates from different locales. One canine and 2 human isolates of serogroup O6 closely resembled archetypal human pyelonephritis isolate 536 (O6:K15:H31), according to papA sequence and VF-serotype profile. These findings support the hypothesis that certain pathogenic lineages of E. coli cause disease in both humans and animals and that humans may acquire pathogenic E. coli from domestic pets.

Animals↗

PubMed1995

DNA profiling of banana and plantain cultivars using random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers.

Polymerase chain reaction (PCR) amplification of genomic DNA from 57 Musa cultivars with 60 random 10-mer primers generated 605 polymorphic amplification products which were useful in unambiguous cultivar identifications. Unweighted pair-group method analysis of this data grouped the cultivars into specific clusters depending on their genomic similarities. The diploid ancestral species of cultivated banana and plantains, namely Musa acuminata sp malaccensis, an A genome donor and M. balbisiana, a B genome donor, were farthest apart from each other in the phenogram. The edible fruit yielding cultivars with the genomic constitutions AA, AAA, AB, AAB, ABB, and ABBB grouped in different clusters according to overall genetic homologies. The restriction fragment length polymorphisms (RFLPs) prevalent among the cultivars were studied by hybridization of 19 random genomic clones to blots of HindIII, EcoRI and MspI digests. Cluster analysis of these data on 107 polymorphic alleles resulted in a phenogram comparable to the one obtained with random amplified polymorphic DNA (RAPD) analysis. Two multilocus probes useful in distinguishing all the 57 cultivars analyzed were also identified. The A and B types of cytoplasms in the cultivars were further distinguished by hybridization of heterologous chloroplast DNA probes. Results showed that use of different kinds of molecular markers in gene banks is essential for characterization and classification of germplasm collections.

Base Sequence↗

PubMed1992

Relatedness of penicillin-binding protein 1a genes from different clones of penicillin-resistant Streptococcus pneumoniae isolated in South Africa and Spain.

Penicillin-resistant strains of Streptococcus pneumoniae have been common in South Africa and Spain for several years. Multilocus enzyme electrophoresis identified one clone of capsular type 6B which was prevalent in Spain and another clone of type 23F that was present in both countries. Genes for penicillin-binding proteins (PBPs) in penicillin-resistant strains are often mosaics where parts of the pneumococcal genes are replaced by homologous genes from other species. We have compared the mosaic structures of the PBP 1a genes from the two clones as well as from genetically distinct South African isolates. Four classes of mosaic PBP 1a genes were found that contained blocks of sequences divergent by 6-22% from those of sensitive genes; two classes contained sequences coming from more than one external source. Data are presented showing that the PBP 1a genes from the 23F and the 6B clone are related, and that the two PBP 1a genes from the South African isolates are also related. We suggest that the type 23F clone originated in Spain prior to distribution into other continents.

Bacterial Proteins↗

PubMed2000

Genomic subpopulations within the species Pediococcus acidilactici detected by multilocus typing analysis: relationships between pediocin AcH/PA-1 producing and non-producing strains.

A high degree of genetic polymorphism among P. acidilactici strains was highlighted by a multilocus typing approach analysing several housekeeping genes and by sampling the whole genome using random amplified polymorphic DNA (RAPD) fingerprint analysis performed by using a single primer pedA gene targeted in low-stringency amplification conditions. Restriction fragment length polymorphism of the rpoC, ldhD/L and mle genes, and a modified RAPD analysis, permitted the grouping of Pediococcus acidilactici strains in seven genotypes (I-VII). Genotypic results obtained by analysing housekeeping genes involved in the transcription/translation machinery and in primary metabolism were supported by phylogenetic analysis based on the partial 16S rDNA sequencing of a reference strain of each of the seven clusters obtained. Three of the seven genotypes detected showed relationships with pediocin AcH/PA-1 production and carbohydrate fermentation patterns: all pediocin-producing and sucrose-positive strains were grouped in genotype VII, melibiose-, sucrose- and raffinose-positive strains in genotype VI, and arabinose-positive strains in genotype V.

Bacterial Typing Techniques↗

PubMed1994

Affected sib-pair analysis of the GLUT1 glucose transporter gene locus in non-insulin-dependent diabetes mellitus (NIDDM): evidence for no linkage.

Despite the strong evidence for a major role played by genetic factors in the aetiology of non-insulin-dependent diabetes mellitus (NIDDM), the genes involved are still unknown. Association studies of candidate genes for the inheritance of NIDDM have so far yielded inconclusive results. Some evidence exists for an association between NIDDM and the glucose transporter gene GLUT1, involved in basal glucose transport, although this has not been confirmed. In the present study we have tested the hypothesis of linkage between NIDDM and the GLUT1 gene, using affected sib-pairs. With this method the concordance observed for a given gene marker is compared with that expected under the assumption of no linkage between that marker and the disease. Fifty-four pedigrees (22 Italians and 32 British), for a total of 82 sib-pairs were studied by the affected sib-pair method proposed by Weeks and Lange, using two restriction fragment length polymorphisms (RFLPs) at the GLUT1 locus, the MspI RFLP, at an estimated 0.171 recombination frequency from the GLUT1 gene, and the XbaI RFLP, located within the GLUT1 gene and previously shown to be associated with the disease. Results showed that the MspI marker and NIDDM segregate independently; for the XbaI RFLP, linkage could be shown only if the results were weighted by the allele frequency [f(p) = 1/p], and only in the Italian and the combined (Italian and British) sib-pair groups. Multilocus analysis with both markers was also negative. We conclude that the GLUT1 gene is very unlikely to play a major role in the aetiology of NIDDM, although an accessory role cannot be excluded, and studies of the gene sequence should help to clarify this question.

Chromosome Mapping↗

PubMed1993

Mycobacterium celatum sp. nov.

A new slowly growing nonphotochromogenic Mycobacterium species of clinical importance is described. The biochemical characteristics of this organism were similar to those of Mycobacterium xenopi and members of the Mycobacterium avium complex. However, none of the strains reacted with commercially available genetic probes for the M. avium complex. The strains were resistant to most antituberculosis drugs. Multilocus enzyme electrophoresis revealed two original electrophoretic types, which was suggestive of new species. The strains contained alpha-, keto-, and dicarboxylic mycolates, as determined by thin-layer chromatography. A mycolic acid analysis by high-performance liquid chromatography revealed a chromatographic pattern similar to that of M. xenopi, but distinct from the patterns of previously described Mycobacterium species. Hexadecanoic and tuberculostearic acids were identified as the major cell wall fatty acids by gas-liquid chromatographic analysis; hexacosanoic acid was the major mycolic acid cleavage product, and 2-eicosanol was the major alcohol. Evaluation of the 16S rRNA sequence confirmed the phylogenetic position of the organism among the slowly growing Mycobacterium species. Cultures representing this new species have been deposited in the American Type Culture Collection as strains ATCC 51130 and ATCC 51131T (T = type strain). The name Mycobacterium celatum is proposed.

Bacterial Typing Techniques↗

PubMed1999

Population biology of Streptococcus pneumoniae isolated from oropharyngeal carriage and invasive disease.

The population structure of Streptococcus pneumoniae in a sample of 134 carried antibiotic-susceptible isolates, and 53 resistant and susceptible invasive isolates, was examined using a DNA-based version of multilocus enzyme electrophoresis: multilocus restriction typing (MLRT). This involved RFLP analysis of PCR products generated from nine loci of housekeeping genes located around the pneumococcal chromosome. The combination of alleles at each of the nine loci gave an allelic profile or restriction type (RT). All carried (throat or nasopharyngeal) isolates from children or adults in Oxford and Manchester, UK, and from an HIV-seropositive cohort in Nairobi, Kenya, showed an epidemic population structure. Twelve carried clonal groups, each with different serotypes, were identified at both locations within the UK. Almost all of the carried clones examined (16/17) were found to possess identical RTs or sequence types (STs) to invasive isolates, indicating that frequently carried clones are also associated with cases of invasive disease. As expected from previous studies, the population of 53 invasive, mainly penicillin-resistant, isolates was also found to be at linkage equilibrium. Serotype switching was identified among 14% of RTs that possessed two or more members, or 5.7% of individual isolates within these RTs. In support of a population structure in which there is frequent recombination, there is also clear evidence that the trpA/B locus within pneumococci has evolved by horizontal gene transfer. A non-serotypable isolate from an HIV-seropositive patient in Kenya was clearly genetically distinct from other strains studied, with unique alleles at eight out of nine loci examined. However, it was initially identified as a pneumococcus by a 16S RNA gene probe (Gen-Probe), optochin susceptibility and the presence of pneumolysin and autolysin.

Adolescent↗

PubMed2001

Phylogenetic evidence for horizontal transfer of mutS alleles among naturally occurring Escherichia coli strains.

mutS mutators accelerate the bacterial mutation rate 100- to 1,000-fold and relax the barriers that normally restrict homeologous recombination. These mutators thus afford the opportunity for horizontal exchange of DNA between disparate strains. While much is known regarding the mutS phenotype, the evolutionary structure of the mutS(+) gene in Escherichia coli remains unclear. The physical proximity of mutS to an adjacent polymorphic region of the chromosome suggests that this gene itself may be subject to horizontal transfer and recombination events. To test this notion, a phylogenetic approach was employed that compared gene phylogeny to strain phylogeny, making it possible to identify E. coli strains in which mutS alleles have recombined. Comparison of mutS phylogeny against predicted E. coli "whole-chromosome" phylogenies (derived from multilocus enzyme electrophoresis and mdh sequences) revealed striking levels of phylogenetic discordance among mutS alleles and their respective strains. We interpret these incongruences as signatures of horizontal exchange among mutS alleles. Examination of additional sites surrounding mutS also revealed incongruous distributions compared to E. coli strain phylogeny. This suggests that other regional sequences are equally subject to horizontal transfer, supporting the hypothesis that the 61.5-min mutS-rpoS region is a recombinational hot spot within the E. coli chromosome. Furthermore, these data are consistent with a mechanism for stabilizing adaptive changes promoted by mutS mutators through rescue of defective mutS alleles with wild-type sequences.

Adenosine Triphosphatases↗

PubMed1995

Multiplex genotype determination at a DNA sequence polymorphism cluster in the human immunoglobulin heavy-chain region.

We have developed a method for multilocus genotype determination. The method involves using restriction fragment length polymorphisms (RFLPs) for allele discrimination. If a polymorphism is not an RFLP, it is converted into an RFLP during the polymerase chain reaction (PCR). After amplification and restriction enzyme digestion, samples are analyzed by sequential gel loading during electrophoresis. The efficiency of this method was demonstrated by determining the genotypes of 108 semen samples at seven DNA sequence polymorphic sites identified in the human immunoglobulin heavy-chain variable region. It was shown that more than 1000 PCR products could be easily analyzed per day per investigator. To show the reliability of this method, some of the typing results were confirmed by DNA sequence analysis. By computer simulation, most (98%) polymorphisms were shown to be natural or convertible (by changing 1 bp close to or next to each polymorphic site) RFLPs for the commercially available 4-base cutters.

Alleles↗

PubMed1991

Proving paternity of children with deceased fathers.

Determination of paternity was attempted in the case of three children whose putative fathers are dead using DNA samples of the paternal grandparents. The DNA analyses were performed with both multilocus and single-locus probes which resolve highly polymorphic areas of human genome. The results were conclusive with both types of probes and facilitated, for example, the exclusion of the brother of the putative father. The evidence for true paternity obtained with DNA analyses can be considered reliable in this type of "indirect" paternity in which tests based on protein polymorphism are inconclusive.

Blood Group Antigens↗

PubMed2000

Genetic diversity analysis of Rhodothermus reflects geographical origin of the isolates.

The genetic diversity and relationships of 81 Rhodothermus isolates from different geothermal environments in Iceland were examined by analysis of electrophoretically demonstrable allelic variation of 13 genes encoding enzymes. All the enzymes were polymorphic. A total of 71 distinctive multilocus genotypes (electrophoretic types, ETs) were identified. The mean genetic diversity per locus (H1) was 0.586. The relatively high genetic variance observed within Rhodothermus isolates from different locations is most likely the result of genetic changes occurring independently in the locations studied. A high Gst value (0.284) indicates that a considerable part of the variance observed is due to differences between locations. Cluster analysis revealed two major groups of ET clusters diverging at a genetic distance of 0.75, reflecting strongly the geographic origin of isolates. Estimation of the association index (I(A)) indicates that Rhodothermus marinus is a clonal species in which recombination events occur rarely. Partial or whole sequencing of the 16S rRNA genes of Rhodothermus isolates grouping at genetic distance of 0.40 confirmed that all the isolates belonged to the species Rhodothermus marinus. The results of this study confirm that, despite phylogenetic and phenotypic similarity, genetic diversity within Rhodothermus marinus is quite high.

Alleles↗

PubMed1994

Refinement of the DNA marker maps of mouse chromosome 12.

To refine the linkage map of mouse Chromosome (Chr) 12 and to define better the homology relationships between it and human chrs 2p and 14q, nine new anonymous DNA markers of Chr 12 were identified, and mouse loci homologous to the human D14S17, CHGA, HSPA2, RRM2, TPO, and ZFP50 ("KUP") genes were defined. The inheritance of DNA variants associated with these markers was followed in progeny of a reciprocal backcross between the C57BL/6J and SWR/J laboratory mouse strains and in recombinant inbred (RI) strains of mice. These data, combined with results of previous analyses of the backcross, allowed the construction of a 22-marker multilocus linkage map that spanned 58 cM. Use of this map to anchor the RI typing data collected in this and previous studies allowed the construction of a 79-marker map that spanned 66 cM and the identification of a framework of unambiguously ordered, extensively typed markers that should facilitate the use of RI mice in testing new markers for possible linkage to Chr 12.

Animals↗

PubMed1993

A novel superantigen isolated from pathogenic strains of Streptococcus pyogenes with aminoterminal homology to staphylococcal enterotoxins B and C.

Streptococcus pyogenes (group A Streptococcus) has re-emerged in recent years as a cause of severe human disease. Because extracellular products are involved in streptococcal pathogenesis, we explored the possibility that a disease isolate expresses an uncharacterized superantigen. We screened culture supernatants for superantigen activity with a major histocompatibility complex class II-dependent T cell proliferation assay. Initial fractionation with red dye A chromatography indicated production of a class II-dependent T cell mitogen by a toxic shock-like syndrome (TSLS) strain. The amino terminus of the purified streptococcal superantigen was more homologous to the amino termini of staphylococcal enterotoxins B, C1, and C3 (SEB, SEC1, and SEC3), than to those of pyrogenic exotoxins A, B, C or other streptococcal toxins. The molecule, designated SSA, had the same pattern of class II isotype usage as SEB in T cell proliferation assays. However, it differed in its pattern of human T cell activation, as measured by quantitative polymerase chain reaction with V beta-specific primers. SSA activated human T cells that express V beta 1, 3, 15 with a minor increase of V beta 5.2-bearing cells, whereas SEB activated V beta 3, 12, 15, and 17-bearing T cells. Immunoblot analysis of 75 disease isolates from several localities detected SSA production only in group A streptococci, and found that SSA is apparently confined to only three clonal lineages as defined by multilocus enzyme electrophoresis typing. Isolates of one of these lineages, (electrophoretic type 2) are strongly associated with TSLS. The data identify SSA as a novel streptococcal superantigen that appears to be more related structurally to staphylococcal enterotoxins than to streptococcal exotoxins. Because abundant SSA production is apparently confined to only three streptococcal clonal lineages, the data also suggest that the SSA gene has only recently been acquired by S. pyogenes.

Amino Acid Sequence↗