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PubMed1994

Evaluation of 13 short tandem repeat loci for use in personal identification applications.

Personal identification by using DNA typing methodologies has been an issue in the popular and scientific press for several years. We present a PCR-based DNA-typing method using 13 unlinked short tandem repeat (STR) loci. Validation of the loci and methodology has been performed to meet standards set by the forensic community and the accrediting organization for parentage testing. Extensive statistical analysis has addressed the issues surrounding the presentation of "match" statistics. We have found STR loci to provide a rapid, sensitive, and reliable method of DNA typing for parentage testing, forensic identification, and medical diagnostics. Valid statistical analysis is generally simpler than similar analysis of RFLP-VNTR results and provides powerful statistical evidence of the low frequency of random multilocus genotype matching.

Alleles↗
PubMed2004

spa typing method for discriminating among Staphylococcus aureus isolates: implications for use of a single marker to detect genetic micro- and macrovariation.

Strain typing of microbial pathogens has two major aims: (i). to index genetic microvariation for use in outbreak investigations and (ii). to index genetic macrovariation for use in phylogenetic and population-based analyses. Until now, there has been no clear indication that one genetic marker can efficiently be used for both purposes. Previously, we had shown that DNA sequence analysis of the protein A gene variable repeat region (spa typing) provides a rapid and accurate method to discriminate Staphylococcus aureus outbreak isolates from those deemed epidemiologically unrelated. Here, using the hypothesis that the genetic macrovariation within a low-level recombinogenic species would accurately be characterized by a single-locus marker, we tested whether spa typing could congruently index the extensive genetic variation detected by a whole-genome DNA microarray in a collection of 36 isolates, which was recovered from 10 countries on four continents over a period of four decades, that is representative of the breadth of diversity within S. aureus. Using spa and coa typing, pulsed-field gel electrophoresis (PFGE), and microarray and multilocus enzyme electrophoresis (MLEE) data in molecular epidemiologic and evolutionary analyses, we determined that S. aureus likely has a primarily clonal population structure and that spa typing can singly index genetic variation with 88% direct concordance with the microarray and can correctly assign isolates to phylogenetic lineages. spa typing performed better than MLEE, PFGE, and coa typing in discriminatory power and in the degree of agreement with the microarray at various phylogenetic depths. This study showed that genetic analysis of the repeat region of protein A comprehensively characterizes both micro- and macrovariation in the primarily clonal population structure of S. aureus.

Antigens, Bacterial↗
PubMed1992

Molecular evolutionary genetics of the cattle-adapted serovar Salmonella dublin.

An electrophoretic analysis of allelic variation at 24 enzyme loci among 170 isolates of the serovar Salmonella dublin (serotype 1,9,12[Vi]:g,p:-) identified three electrophoretic types (Du 1, Du 3, and Du 4), marking three closely related clones, one of which (Du 1) is globally distributed and was represented by 95% of the randomly selected isolates. All but 1 of 114 nonmotile isolates of serotype 1,9,12:-:- recovered from cattle and swine in the United States were genotypically Du 1. The virulence capsular polysaccharide (Vi antigen) is confined to clone Du 3, which apparently is limited in distribution to France and Great Britain. For all 29 isolates of Du 3, positive signals were detected when genomic DNA was hybridized with a probe specific for the ViaB region, which contains the structurally determinant genes for the Vi antigen; and 23 of these isolates had been serologically typed as Vi positive. In contrast, all 30 isolates of Du 1 tested with the ViaB probe were negative. These findings strongly suggest that the ViaB genes were recently acquired by S. dublin via horizontal transfer and additive recombination. The clones of S. dublin are closely similar to the globally predominant clone (En 1) of Salmonella enteritidis (serotype 1,9,12:g,m:-) in both multilocus enzyme genotype and nucleotide sequence of the fliC gene encoding phase 1 flagellin. Comparative sequencing of fliC has revealed the molecular genetic basis for expression of the p and m flagellar epitopes by which these serovars are distinguished in the Kauffmann-White serological scheme of classification.

Alleles↗
PubMed2005

Microsatellite marker analysis as a typing system for Candida glabrata.

Candida glabrata is one of the most important causes of nosocomial fungal infection. We investigated, using a multiplex PCR, three polymorphic microsatellite markers, RPM2, MTI, and ERG3, in order to obtain a rapid genotyping method for C. glabrata. One set of primers was designed for each locus, and one primer of each set was dye labeled to read PCR signals using an automatic sequencer. Eight reference strains including other Candida species and 138 independent C. glabrata clinical isolates were tested. The clinical isolates were collected from different anatomical sites of adult patients either hospitalized in different wards of two different hospitals or not hospitalized. Since C. glabrata is haploid, one single PCR product for each PCR set was obtained and assigned to an allele. The numbers of different alleles were 5, 7, and 15 for the RPM2, MTI, and ERG3 loci, respectively. The number of allelic associations was 21, leading to a discriminatory power of 0.84. The markers were stable after 25 subcultures, and the amplifications were specific for C. glabrata. A factorial correspondence analysis did not indicate any correlation between the 21 multilocus genotypes and the clinical data (source, sex, ward, anatomical sites). Microsatellite marker analysis is a rapid and reliable technique to investigate clinical issues concerning C. glabrata. However, its discriminatory power should be improved by testing other polymorphic microsatellite loci.

Adult↗
PubMed1996

Molecular genetic relationships of the salmonellae.

A multilocus enzyme electrophoresis analysis of 96 strains of the salmonellae distinguished 80 electrophoretic types (ETs) and placed them in eight groups, seven of which correspond precisely to the seven taxonomic groups (I, II, IIIa, IIIb, IV, V, and VI) previously defined on the basis of biotype and genomic DNA hybridization. In addition, multilocus enzyme electrophoresis identified an eighth distinctive group (designated VII) composed of five strains that had been assigned to group IV on the basis of biotype. An analysis of variation in the combined nucleotide sequences of five housekeeping genes among 16 strains representing all eight groups yielded estimates of overall genetic relationships that are fully consistent with those indicated by DNA hybridization. However, the nucleotide sequences of seven invasion genes (inv/spa) in the strains of group VII were closely similar to those of strains of group IV. These findings are interpreted as evidence that group VII represents an old, differentiated lineage to which one or more large parts of the chromosomal genome of the group IV lineage, including the 40-kb segment on which the invasion genes are located, have been horizontally transferred. All lines of molecular genetic evidence indicate that group V is very strongly differentiated from all other groups, thus supporting its current taxonomic treatment as a species, Salmonella bongori, separate from S. enterica. The Salmonella Reference Collection C, composed of the 16 strains used in DNA sequence studies, has been established for research on variation in natural populations.

Alleles↗
PubMed2002

Afa, a diffuse adherence fibrillar adhesin associated with enteropathogenic Escherichia coli.

O55 is one of the most frequent enteropathogenic Escherichia coli (EPEC) O serogroups implicated in infantile diarrhea in developing countries. Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA). Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa. An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H(-) ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic E. coli. Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures. This is the first characterization and demonstration of an Afa adhesin associated with EPEC.

Adhesins, Escherichia coli↗
PubMed1998

Genetic diversity among strains of Moraxella catarrhalis: analysis using multiple DNA probes and a single-locus PCR-restriction fragment length polymorphism method.

Moraxella (Branhamella) catarrhalis, a causative agent of otitis media, sinusitis, and exacerbation of bronchitis, has acquired widespread ability to produce beta-lactamase and can be nosocomially transmitted. The typing methods used in epidemiological analyses of M. catarrhalis are not optimal for genetic analyses. Two methods, a multiple-locus Southern blot (SB) method and a single-locus PCR-restriction fragment length polymorphism (RFLP) method, were developed and used to assess genetic diversity and potential clinical and geographic relationships in M. catarrhalis. Nine randomly cloned M. catarrhalis DNA fragments were used as probes of SBs containing DNA from 54 geographically and clinically diverse strains. For comparison, a PCR-RFLP method was developed as a quick, inexpensive, and discriminating alternative. A highly variable 3.7-kb genomic region (M46) was cloned and sequenced, and 3.5 kb of the cloned DNA was targeted for PCR amplification. DNAs from the 54 strains were subjected to PCR-RFLP. SB analysis distinguished all strains that had no apparent epidemiological linkage (40 of 54), and PCR-RFLP distinguished fewer strains (21 of 54). Epidemiologically linked strains appeared genetically identical by both methods. PCR-RFLP was compared to pulsed-field gel electrophoresis (PFGE) for 8 of the 54 strains and 23 additional strains. PCR-RFLP distinguished fewer strains than PFGE typing (16 of 31 versus 20 of 31 strains), but PCR-RFLP was more useful for inferring interstrain relatedness. Separate cluster analyses of multilocus SB and single locus PCR-RFLP data showed high genetic diversity within and across geographic locations and clinical presentations. The resultant dendrograms were not entirely concordant, but both methods often gave similar strain clusters at the terminal branches. High genetic diversity, nonconcordance of cluster analyses from different genetic loci, and shared genotypes among epidemiologically linked strains support a hypothesis of high recombination relative to spread of clones. Single-locus PCR-RFLP may be suitable for short-term epidemiological studies, but the SB data demonstrate that greater strain discrimination may be obtained by sampling variation at multiple genomic sites.

Adult↗
PubMed2006

Delineation of Borrelia burgdorferi sensu lato species by multilocus sequence analysis and confirmation of the delineation of Borrelia spielmanii sp. nov.

To evaluate multilocus sequence analysis (MLSA) for taxonomic purposes in the delineation of species within Borrelia burgdorferi sensu lato, seven relevant loci of various strains for which extensive DNA-DNA reassociation data were available were sequenced. MLSA delineation proved to be fully concordant with conventional methods. Our analysis confirmed the delineation of a novel species, Borrelia spielmanii sp. nov., previously known as 'Borrelia spielmani' Richter et al. 2004, with strain PC-Eq17N5T (=DSM 16813T = CIP 108855T) as the type strain.

Bacterial Typing Techniques↗
PubMed2004

Antigen genes for molecular epidemiology of leishmaniasis: polymorphism of cysteine proteinase B and surface metalloprotease glycoprotein 63 in the Leishmania donovani complex.

BACKGROUND: Efficient monitoring of endemic and resurgent visceral leishmaniasis (VL) requires discriminatory molecular tools that allow direct characterization of etiological agents (i.e., the Leishmania donovani complex) in host tissues. This characterization is possible through restriction fragment-length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified sequences (PCR-RFLP). METHODS: We present 2 new PCR-RFLP assays that target the gene locus of cysteine proteinase B (cpb), an important Leishmania antigen. The assays were applied to the characterization of 15 reference strains of the L. donovani complex, and their discriminatory power was compared with that of PCR-RFLP analysis of the gp63 gene, another Leishmania antigen, and with that of multilocus enzyme electrophoresis (MLEE), which is the reference standard for parasite typing. RESULTS: Restriction patterns of the cpb locus were polymorphic, but less so than gp63 patterns. When data for both loci were combined, differences between PCR-RFLP and MLEE results were encountered. Antigen gene analysis was more discriminatory and supported a different classification of parasites, one that fitted with their geographic origin. PCR-RFLP analysis of cpb also allowed direct genotyping of parasites in bone marrow aspirate and venous blood samples obtained from patients with VL. CONCLUSION: Antigen genes constitute valid targets for PCR-based Leishmania typing without the need for isolation of parasites.

Animals↗
PubMed1999

Methods for the differentiation of microorganisms.

Advances in analytical and diagnostic assays based on novel nucleic acid analyses techniques have revolutionized the application of molecular differentiation of microorganisms. Phenotypic typing schemes are now broadly supplemented by new genotyping methods which allow a more refined and detailed differentiation of closely related microorganisms, bacterial strains, isolates and pathogens on the DNA level. Bio-, sero- and phagetyping, antibiotic susceptibility tests, immunoblotting as well as multilocus enzyme- or polyacrylamide gel electrophoresis are now supported by the analysis of plasmid or chromosomal DNA restriction profiles, ribotyping, pulsed-field gel electrophoresis and polymerase- or ligase-chain reaction-based methods or direct sequencing technique to differentiate microorganisms. Some of these molecular techniques are also used in the field of virology to analyse and differentiate closely related sub- or genotypes. Few examples for the analysis and investigation of these usually small genomes will also be given.

Genotype↗
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↗
PubMed2006

Multilocus genotyping of Cryptosporidium hominis associated with diarrhea outbreak in a day care unit in São Paulo.

UNLABELLED: A number of species of Cryptosporidium are associated with diarrhea worldwide. Little data exists regarding the genotypes and species of Cryptosporidium associated with cases of infections in Brazil. PURPOSE: In the present study, we ascertained by molecular methods the species and the genotype of Cryptosporidium sp from a diarrhea outbreak diagnosed in a day care at the Hospital Clínicas, São Paulo University Medical School. MATERIALS AND METHODS: Specific identification and typing of the isolates associated with the outbreak was done by DNA sequencing analysis of fragments amplified by polymerase chain reaction (PCR) from 3 different Cryptosporidium loci: the SSUrRNA coding region, the Cryptosporidium oocyst wall protein (COWP) gene, and the microsatellite locus 1 (ML1), a tandem GAG-trinucleotide repeat containing substitutions that differentiate the genotypes of Cryptosporidium parvum and Cryptosporidium hominis. RESULTS: A total of 29 positive samples from the outbreak were studied by the molecular methods described. Our study revealed the presence of a single genotype of Cryptosporidium hominis in all samples. CONCLUSION: The molecular analysis reinforced the hypothesis that the transmission of Cryptosporidium hominis during the period the samples were collected occurred in an outbreak pattern, possibly by person-to-person contact through the fecal-oral route. As far as we know, this is the first time that molecular tools have been used to identify the species and the genotype of isolates showing the presence of the ML1 genotype in samples from Brazilian patients.

Animals↗
PubMed2002

Strain typing in Leishmania donovani by using sequence-confirmed amplified region analysis.

To differentiate strains of Leishmania donovani, allelic markers at the DNA level were developed by sequence-confirmed amplified region analysis (SCAR). Homologous fragments from different strains of L. donovani were amplified by PCR using random primers and subsequently screened for single-strand conformation polymorphisms. Direct sequencing revealed 55 sequence polymorphisms in eight co-dominant DNA markers; 38 of them were single point mutations. Heterozygosity was evident for 69% and fixed heterozygosity for 25% of all polymorphisms. At most polymorphic sites one of the segregation genotypes was missing. Nineteen unique multilocus genotypes were identified among 29 strains of L. donovani. One genotype was represented by eight Sudanese strains; also two strains from Sudan as well as two strains from Kenya, respectively, shared identical genotypes. All other strains had individual multilocus genotypes. Calculation of genetic distances showed a correlation between multilocus genotypes and the geographical origin of these strains. African strains were found in one well-supported cluster with Kenyan and Sudanese strains clearly separated. SCAR markers seem to represent a random sample of neutral genetic variation present in natural populations. They are co-dominant because they can detect all possible allele combinations in a diploid organism and may, therefore, be very useful for population genetic analysis in Leishmania.

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↗
PubMed2005

Multilocus DNA sequence comparisons rapidly identify pathogenic molds.

The increasing incidence of opportunistic fungal infections necessitates rapid and accurate identification of the associated fungi to facilitate optimal patient treatment. Traditional phenotype-based identification methods utilized in clinical laboratories rely on the production and recognition of reproductive structures, making identification difficult or impossible when these structures are not observed. We hypothesized that DNA sequence analysis of multiple loci is useful for rapidly identifying medically important molds. Our study included the analysis of the D1/D2 hypervariable region of the 28S ribosomal gene and the internal transcribed spacer (ITS) regions 1 and 2 of the rRNA operon. Two hundred one strains, including 143 clinical isolates and 58 reference and type strains, representing 43 recognized species and one possible new species, were examined. We generated a phenotypically validated database of 118 diagnostic alleles. DNA length polymorphisms detected among ITS1 and ITS2 PCR products can differentiate 20 of 33 species of molds tested, and ITS DNA sequence analysis permits identification of all species tested. For 42 of 44 species tested, conspecific strains displayed >99% sequence identity at ITS1 and ITS2; sequevars were detected in two species. For all 44 species, identifications by genotypic and traditional phenotypic methods were 100% concordant. Because dendrograms based on ITS sequence analysis are similar in topology to 28S-based trees, we conclude that ITS sequences provide phylogenetically valid information and can be utilized to identify clinically important molds. Additionally, this phenotypically validated database of ITS sequences will be useful for identifying new species of pathogenic molds.

DNA, Fungal↗