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Results for “Multilocus sequence typing”

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PubMed2003

Typing of nonencapsulated haemophilus strains by repetitive-element sequence-based PCR using intergenic dyad sequences.

Intergenic dyad sequences (IDS) are short repeated elements that have been described for several Haemophilus genomes and for only two other bacterial genera. We developed a repetitive-element sequence-based PCR using an IDS-specific primer as a typing method (IDS-PCR) for nonencapsulated Haemophilus strains and compared this technique with pulsed-field gel electrophoresis (PFGE) of DNA restricted with SmaI. IDS-PCR was rapid, easy to perform, and reproducible, with a high discriminatory capacity for nontypeable Haemophilus influenzae (NTHI) strains. The 69 NTHI strains tested generated 65 different banding patterns. Epidemiologically related strains gave similar or identical fingerprints, and all of the unrelated strains except two showed different patterns. These results were in agreement with those obtained by PFGE. For 20 genital strains usually identified as being biotype IV NTHI and belonging to a cryptic genospecies of Haemophilus with remarkable genetic homogeneity, four bands were significantly present and six bands were significantly absent from the fingerprints. The 20 strains were gathered in 11 closely related profiles, whereas PFGE provided no band when DNA was treated with SmaI. IDS-PCR improved the differentiation previously obtained within this species by ribotyping and multilocus enzyme electrophoresis. Our findings suggest that IDS-PCR is a rapid, reliable, and discriminatory method for typing NTHI strains and is currently the most efficient method for distinguishing strains within the cryptic genospecies of HAEMOPHILUS:

Base Sequence↗

PubMed2001

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: population genetics, human serologic response, and gene transcription.

Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase-PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research.

Amino Acid Sequence↗

PubMed1992

Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms.

Staphylocoagulase, a major phenotypic determinant of Staphylococcus aureus, exists in multiple allelic forms, in part because of the existence of gene variants within the 3'-end coding region. This region contains a series of repeating 81-bp DNA sequences which differ both in the number of tandem repeats and the location of AluI restriction sites among different isolates. Utilizing this finding, we developed a novel typing method for S. aureus based on polymerase chain reaction amplification of the variable region of the coagulase gene followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP). Among 30 S. aureus isolates studied initially, a total of 10 distinct RFLP patterns were observed. There was excellent correlation of the RFLP patterns with typing of these isolates by multilocus enzyme electrophoresis at 20 chromosomal loci. This coagulase RFLP method was used to analyze an additional 39 S. aureus isolates and successfully traced the source of an outbreak of methicillin-resistant S. aureus infections at a local hospital.

Bacterial Typing Techniques↗

PubMed1989

Distribution of the P-associated-pilus (pap) region among Escherichia coli from natural sources: evidence for horizontal gene transfer.

Variation in chromosomal DNA in Escherichia coli was studied with probes specific for the P-associated-pilus (pap) region. The presence of DNA homologous to pap was determined by dot blots. Variation in the number of copies of pap and in the organization of internal and flanking sequences was determined by Southern blot hybridization. The 229 strains studied were also classified by O:K:H serotyping and multilocus enzyme electrophoresis. There was considerable heterogeneity in the presence of pap and distribution of pap-homologous DNA in these E. coli strains from natural sources. In general, there was less variation in pap among strains of the same specific O:K:H serotype and enzyme electrophoretic type than among random isolates. There were, however, E. coli strains identified as members of the same clone by O:K:H serotyping and enzyme electrophoresis that were pap positive and pap negative or had different Southern blot patterns for the pap probes (pap type). There were also isolates of the same pap type that differed in two of three O:K:H serotype antigens and the majority of enzymes that determined their enzyme electrophoretic type. These latter two observations were interpreted as evidence for the horizontal (infectious) transfer of the pap-homologous sequences among clones of E. coli.

Bacterial Adhesion↗

PubMed2004

[The effect of routine vaccination in the Czech Republic on the incidence of invasive diseases caused by Haemophilus influenzae b].

OBJECTIVES: The paper is an analysis of the results of a five-year surveillance programme in the Czech Republic (1999-2003); it evaluates the efficacy of routine Hib vaccination that started in July 2001. MATERIAL AND METHODS: Morbidity due to Hib was explored in a surveillance programme, which since 2002 also included the investigation of Hib vaccine failure. Hib identification was carried out with standard methods, serotypes were verified using PCR, biotyping was carried out in all strains and in selected strains also multilocus sequential typing. RESULTS: In the years 1999-2003 invasive Hib disease presented mostly as meningitis, followed by epiglottitis. Mortality due to an invasive Hib disease was in the years 1999-2003 2.3 %. Among the Hib strains isolated in invasive disorders predominated the biotype I and the sequence type ST-6. CONCLUSIONS: Following the introduction of routine Hib vaccination in the Czech Republic there was an overall drop in morbidity due to Hib invasive disorders. This was most obvious in a decrease in the morbidity of Hib meningitis and in the vaccinated age group. Two years after the introduction of routine Hib vaccination morbidity dropped by 81 % in children aged 0 to 1 year. In higher age groups there was no change in the number of invasive Hib disease. Neither was there an increase in "non-b" haemophilus invasive disorders. Failure of Hib vaccination is a rare occurence.

Czech Republic↗

PubMed1994

Genetic Structure of Rhizobium leguminosarum biovar trifolii and viciae Populations Found in Two Oregon Soils under Different Plant Communities.

An investigation was carried out to determine the genetic structure in soil populations of Rhizobium leguminosarum bv. trifolii and viciae at each of two Oregon sites (A and C) that were 1 km apart. Although the soils were similar, the plant communities were quite different because grazing by domestic animals had been allowed (site A) or prevented (site C). Analysis of allelic variation at 13 enzyme-encoding loci by multilocus enzyme electrophoresis delineated 202 chromosomal types (ETs) among a total of 456 isolates representing two populations of R. leguminosarum bv. trifolii (AT and CT) and two populations of R. leguminosarum bv. viciae (AV and CV). Regardless of their site of origin or biovar affiliation, isolates of the same ET were confirmed to be more closely related to each other than to isolates of other ETs by repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences and the PCR technique. Despite the wide range in densities of the Rhizobium populations (<10 to >10/g of soil), their overall genetic diversities were similar (mean genetic diversity, 0.45 to 0.51), indicating that low-density populations of soil-borne bacterial species are not necessarily of little genetic diversity. Linkage disequilibrium analysis revealed significant multilocus structure (nonrandom associations of alleles) within each of the four populations. From a combination of cluster and linkage disequilibrium analyses, a total of eight distinct groups of ETs were defined in the four populations. Two groups (I and III) contributed significant numbers of ETs and isolates to each population. The two populations of R. leguminosarum bv. viciae (AV and CV) exhibited similar genetic structures despite existing at different densities, in different plant communities, and in the presence (CV) or absence (AV) of their local Vicia hosts. In contrast, total linkage disequilibrium was partitioned differently in two biovar populations occupying the same soil (AV and AT), with disequilibrium in the latter being due entirely to the presence of group V.

Journal Article↗

PubMed2001

Bacillus sonorensis sp. nov., a close relative of Bacillus licheniformis, isolated from soil in the Sonoran Desert, Arizona.

Eight Bacillus strains isolated from Sonoran Desert soil were shown to belong to a previously unidentified species, for which the name Bacillus sonorensis sp. nov. is proposed. The type strain is strain L87-10T (= NRRL B-23154T). On the basis of phenotypic and genetic data, B. sonorensis is most closely related to Bacillus licheniformis. B. sonorensis can be distinguished from B. licheniformis by salt tolerance, pigmentation, multilocus enzyme electrophoresis, reassociation of genomic DNA and sequence differences in protein-coding genes and 16S rRNA.

Arizona↗

PubMed2006

HOOF prints: Brucella strain typing by PCR amplification of multilocus tandem-repeat polymorphisms.

A critical component of limiting bacterial disease outbreaks is the tracing of the infection to the index source, which can be facilitated by using a highly discriminating bacterial identification system that will reliably identify genetically related bacterial populations. For pathogenic bacteria with highly conserved genomes, such as the zoonotic pathogen Brucella, finding distinguishing markers or traits for strain identification is challenging. This chapter describes a relatively new procedure for identifying Brucella strains. The procedure, which is called "HOOF prints" (hypervariable octameic oligonucleotide fingerprints), is based on high levels of polymorphism observed at several genomic loci in the Brucella genomes that contain small tandemly repeated deoxyribonucleic sequences. The technique described is designed for medium- to high-throughput analyses. However, the method described can be modified to characterize fewer samples.

Animals↗

PubMed1999

Genetic diversity in the yeast species Malassezia pachydermatis analysed by multilocus enzyme electrophoresis.

Fifty-two strains of the yeast species Malassezia pachydermatis were analysed by multilocus enzyme electrophoresis. M. pachydermatis appeared to be genetically heterogeneous. A total of 27 electrophoretic types were identified that could be divided into five distinct groups with different host specificities. The diversity revealed by this electrophoretic method matched remarkably well the reported genetic variability obtained by comparing large subunit rRNA sequences. This study also suggests that genetic exchanges can occur in the anamorphic species M. pachydermatis.

Animals↗

PubMed1998

Sequence polymorphism in the beta-tubulin gene reveals heterogeneous and variable population structures in Cryptosporidium parvum.

Restriction fragment length polymorphism (RFLP) analysis of isolates of Cryptosporidium parvum has revealed two subgroups, termed H and C. The limited resolution of the RFLP method precludes an in-depth study of the genetic structure of C. parvum populations. Published C. parvum restriction polymorphisms lie within protein-coding regions known to be more homogeneous than noncoding sequences. To better assess the degrees of heterogeneity between and within C. parvum isolates, sequence polymorphism in the beta-tubulin intron, the only C. parvum intron described to date, was investigated. In contrast to the two genotypes distinguished by multilocus RFLP, several alleles were detected by sequence and RFLP analysis of the beta-tubulin intron and adjacent exon 2. Isolates carrying different beta-tubulin alleles were found. Significantly, one of the beta-tubulin alleles present in two geographically unrelated isolates combined features of C- and H-type isolates, suggesting that it might have arisen from a recombination event. A comparison of multiple samples of a calf-propagated laboratory isolate showed that the ratio of different beta-tubulin alleles fluctuated during serial passage.

Animals↗

PubMed1992

Evidence consistent with horizontal transfer of the gene (emm12) encoding serotype M12 protein between group A and group G pathogenic streptococci.

Human isolates of Lancefield group G streptococci harbor sequences homologous with the structural gene (emm) encoding M protein, a major virulence factor in Streptococcus pyogenes (a group A Streptococcus species). We used DNA-DNA hybridization, restriction endonuclease chromosomal profiling, and multilocus enzyme electrophoresis to examine genetic relationships between group A and group G streptococcal strains expressing homologous serologic type 12 M (M12) protein. All M12 group A strains studied had very similar restriction endonuclease genomic profiles and multilocus enzyme genotypes. In contrast, the restriction enzyme genomic profile and multilocus enzyme genotype of the M12 group G strain CS140 were strikingly different from those characterizing the M12 group A organisms. DNA-DNA hybridization studies revealed, on average, 57% genomic similarity between the M12 group A and group G strains. Taken together, our data demonstrate that a gene encoding M12 protein occurs in two highly divergent chromosomal backgrounds, a result suggesting that an episode of horizontal gene transfer and recombination has occurred between two streptococcal lineages.

Antigens, Bacterial↗

PubMed2006

Reclassification of Lactobacillus amylophilus LMG 11400 and NRRL B-4435 as Lactobacillus amylotrophicus sp. nov.

The taxonomic position of six Lactobacillus amylophilus strains isolated from swine waste-corn fermentations was reinvestigated. All strains were included in a multilocus sequence analysis (MLSA) study for species identification of Lactobacillus using the genes encoding the phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA). Partial pheS and rpoA gene sequences showed that strains LMG 11400 and NRRL B-4435 represent a separate lineage that is distantly related to the type strain of L. amylophilus, LMG 6900T, and to three other strains of the species. The MLSA data showed that the two strains LMG 11400 and NRRL B-4435 constituted a distinct cluster, sharing 100% pheS and rpoA gene sequence similarity. The other reference strains clustered together with the type strain of L. amylophilus, LMG 6900T, and were clearly differentiated from strains LMG 11400 and NRRL B-4435 (80 and 89% pheS and rpoA gene sequence similarity, respectively). The 16S rRNA gene sequences of the latter two strains are 100% identical, with the nearest phylogenetic neighbour L. amylophilus LMG 6900T showing only 97.2% 16S rRNA gene sequence similarity. Further polyphasic taxonomic study based on whole-cell protein fingerprinting, DNA-DNA hybridization and biochemical features demonstrated that the two strains represent a single, novel Lactobacillus species, for which the name Lactobacillus amylotrophicus sp. nov. is proposed. The type strain is LMG 11400T (=NRRL B-4436T=DSM 20534T).

Animal Husbandry↗

PubMed2004

Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes: a review.

Listeria monocytogenes is an important food-borne pathogen and is widely tested for in food, environmental and clinical samples. Identification traditionally involved culture methods based on selective enrichment and plating followed by the characterization of Listeria spp. based on colony morphology, sugar fermentation and haemolytic properties. These methods are the gold standard; but they are lengthy and may not be suitable for testing of foods with short shelf lives. As a result more rapid tests were developed based on antibodies (ELISA) or molecular techniques (PCR or DNA hybridization). While these tests possess equal sensitivity, they are rapid and allow testing to be completed within 48 h. More recently, molecular methods were developed that target RNA rather than DNA, such as RT-PCR, real time PCR or nucleic acid based sequence amplification (NASBA). These tests not only provide a measure of cell viability but they can also be used for quantitative analysis. In addition, a variety of tests are available for sub-species characterization, which are particularly useful in epidemiological investigations. Early typing methods differentiated isolates based on phenotypic markers, such as multilocus enzyme electrophoresis, phage typing and serotyping. These phenotypic typing methods are being replaced by molecular tests, which reflect genetic relationships between isolates and are more accurate. These new methods are currently mainly used in research but their considerable potential for routine testing in the future cannot be overlooked.

Animals↗

PubMed1998

A microsatellite-based multilocus phylogeny of the Drosophila melanogaster species complex.

Uncovering the genealogy of closely related species remains a major challenge for phylogenetic reconstruction. It is unlikely that the phylogeny of a single gene will represent the phylogeny of a species as a whole [1], but DNA sequence data across a large number of loci can be combined in order to obtain a consensus tree [2]. Long sequences are needed, however, to minimize the effect of (infrequent) base substitutions, and sufficient individuals must be sequenced per species to account for intraspecific polymorphisms, an overwhelming task using current DNA sequencing technology. By contrast, microsatellites are easy to type [3], allowing the analysis of many loci in multiple individuals. Despite their successful use in mapping [4,5], behavioural ecology [6] and population genetics [7], their usefulness for the phylogenetic reconstruction of closely related taxa has never been demonstrated, even though microsatellites are often conserved across species [8-10]. One drawback to microsatellite use is their high mutation rate (10(-4)-10(-2)), combined with an incomplete understanding of their mutation patterns. Many microsatellites are available for Drosophila melanogaster, and they are distributed throughout the genome [11]. Most can be amplified in the D. melanogaster species complex [12,13] and have low mutation rates [14, 15]. We show that microsatellite-specific distance measurements [16] correlate with other multilocus distances, such as those obtained from DNA-DNA hybridization data. Thus microsatellites may provide an ideal tool for building multilocus phylogenies. Our phylogenetic reconstruction of the D. melanogaster complex provides strong evidence that D. sechellia arose first, followed by a split between D. simulans and D. mauritiana.

Animals↗

PubMed2005

Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients.

The aim of this study was to evaluate molecular and phenotypic methods for the identification of nonhemolytic streptococci. A collection of 148 strains consisting of 115 clinical isolates from cases of infective endocarditis, septicemia, and meningitis and 33 reference strains, including type strains of all relevant Streptococcus species, were examined. Identification was performed by phylogenetic analysis of nucleotide sequences of four housekeeping genes, ddl, gdh, rpoB, and sodA; by PCR analysis of the glucosyltransferase (gtf) gene; and by conventional phenotypic characterization and identification using two commercial kits, Rapid ID 32 STREP and STREPTOGRAM and the associated databases. A phylogenetic tree based on concatenated sequences of the four housekeeping genes allowed unequivocal differentiation of recognized species and was used as the reference. Analysis of single gene sequences revealed deviation clustering in eight strains (5.4%) due to homologous recombination with other species. This was particularly evident in S. sanguinis and in members of the anginosus group of streptococci. The rate of correct identification of the strains by both commercial identification kits was below 50% but varied significantly between species. The most significant problems were observed with S. mitis and S. oralis and 11 Streptococcus species described since 1991. Our data indicate that identification based on multilocus sequence analysis is optimal. As a more practical alternative we recommend identification based on sodA sequences with reference to a comprehensive set of sequences that is available for downloading from our server. An analysis of the species distribution of 107 nonhemolytic streptococci from bacteremic patients showed a predominance of S. oralis and S. anginosus with various underlying infections.

Bacteremia↗

PubMed1995

Vir typing: a long-PCR typing method for group A streptococci.

We have developed a new procedure (Vir typing) for typing Streptococcus pyogenes, by amplifying the entire 5- to 7-kb variable vir regulon by long PCR. The amplified DNA is then cleaved with HaeIII and visualized by ethidium bromide fluorescence after agarose gel electrophoresis. A simple procedure for preparing DNA of sufficiently high quality from 96 samples was employed simultaneously. This DNA was also used to develop a random amplified polymorphic DNA (RAPD) procedure. The discriminatory power of the two DNA-based procedures was compared with previous methods, M typing, and multilocus enzyme electrophoresis. Both procedures were highly discriminatory, but the stoichiometric yield of restriction fragments in Vir typing allows unambiguous interpretation of results.

Bacterial Proteins↗

PubMed2006

Reclassification of Serpulina intermedia and Serpulina murdochii in the genus Brachyspira as Brachyspira intermedia comb. nov. and Brachyspira murdochii comb. nov.

A reassessment was undertaken of published characteristics of the five species of anaerobic intestinal spirochaetes in the genus Brachyspira (Brachyspira aalborgi, Brachyspira alvinipulli, Brachyspira hyodysenteriae, Brachyspira innocens and Brachyspira pilosicoli) and the two species in the genus Serpulina (Serpulina intermedia and Serpulina murdochii). Comparisons were made of published descriptions, including phenotypic properties, grouping by multilocus enzyme electrophoresis, DNA base composition, DNA-DNA relative reassociation values and 16S rRNA gene sequence similarity. On the basis of extensive similarities between all the species, it is proposed to reclassify Serpulina intermedia Stanton et al. 1997 and Serpulina murdochii Stanton et al. 1997 in the genus Brachyspira, as Brachyspira intermedia comb. nov. (type strain ATCC 51140T) and Brachyspira murdochii comb. nov. (type strain ATCC 51284T).

Base Composition↗

PubMed2004

Pulsed-field gel electrophoresis study of Mycobacterium abscessus isolates previously affected by DNA degradation.

DNA degradation (which results in a smear pattern) occurs with almost 50% of Mycobacterium abscessus strains during pulsed-field gel electrophoresis (PFGE). We assessed the potential benefit of using thiourea-containing buffer with M. abscessus by studying 69 isolates not previously typeable by PFGE (i.e., those with a smear pattern). Random (epidemiologically unrelated) isolates that were typeable (no smear pattern) were included as controls. Genomic DNA was digested with DraI, XbaI, and AseI. PFGE gels were run in regular gel buffer with and without 100 muM thiourea. All 69 isolates that generated smear patterns had clear band profiles when the thiourea buffer was used. These isolates were divided into only 30 patterns with DraI, 20 patterns with XbaI, and 20 patterns with AseI. The molecular profiles were all closely or possibly related, and the differences between the isolates ranged from zero to six bands. By multilocus enzyme electrophoresis (MEE), 45 of 53 smear isolates (85%) belonged to two closely related electrophoretic types. These isolates contained at least one enzyme allele seen almost exclusively in this group. Isolates without smear patterns were unaffected by thiourea and produced unrelated PFGE profiles, as well as multiple MEE types. The hsp65 and 16S rRNA gene sequences of the isolates with smear patterns were identical to those of M. abscessus type strain ATCC 19977, which had a nonsmear pattern, suggesting that this clone is a subgroup within M. abscessus. This demonstrates that the inability to type M. abscessus by PFGE is associated with a single clone of organisms.

Bacterial Proteins↗