SEARCH · PubMed Health

Results for “MLST”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources

PubMed2006

Comparison of multilocus sequence typing and Ca3 fingerprinting for molecular subtyping epidemiologically-related clinical isolates of Candida albicans.

Southern hybridization with the complex probe Ca3 is a well established tool for molecular subtyping of Candida albicans. Multilocus sequence typing (MLST) is a DNA sequence-based subtyping method recently applied to C. albicans and shown to have a high degree of intraspecies discriminatory power. However, its utility for studying the molecular epidemiology of sequential isolates from recurrent disease has not been established. We compared Ca3 Southern hybridization and MLST using seven housekeeping genes (CaAAT1a, CaACC1, CaADP1, CaPMI, CaSYA1, CaVPS13, CaZWF1b) for their ability to discriminate among 37 C. albicans isolates from recurrent cases of oropharyngeal candidiasis (OPC) in ten HIV-positive patients from India and the US. Among the 37 isolates, MLST identified 23 distinct genotypes (index of diversity = 97%); Ca3 Southern hybridization identified 21 distinct genotypes (index of diversity = 95%). Both methods clustered isolates into seven genetically-related groups and, with one exception, isolates that were indistinguishable by MLST were indistinguishable or highly related by Ca3 Southern hybridization. These results demonstrate that MLST performs equally well or better compared to Ca3 Southern hybridization for defining genetic-relatedness of sequential C. albicans isolates from recurrent cases of OPC in HIV-positive patients.

Blotting, Southern↗

PubMed2005

The multilocus sequence typing network: mlst.net.

The unambiguous characterization of strains of a pathogen is crucial for addressing questions relating to its epidemiology, population and evolutionary biology. Multilocus sequence typing (MLST), which defines strains from the sequences at seven house-keeping loci, has become the method of choice for molecular typing of many bacterial and fungal pathogens (and non-pathogens), and MLST schemes and strain databases are available for a growing number of prokaryotic and eukaryotic organisms. Sequence data are ideal for strain characterization as they are unambiguous, meaning strains can readily be compared between laboratories via the Internet. Laboratories undertaking MLST can quickly progress from sequencing the seven gene fragments to characterizing their strains and relating them to those submitted by others and to the population as a whole. We provide the gateway to a number of MLST schemes, each of which contain a set of tools for the initial characterization of strains, and methods for relating query strains to other strains of the species, including clustering based on differences in allelic profiles, phylogenetic trees based on concatenated sequences, and a recently developed method (eBURST) for identifying clonal complexes within a species and displaying the overall structure of the population. This network of MLST websites is available at http://www.mlst.net.

Alleles↗

PubMed2006

Identification of host-associated alleles by multilocus sequence typing of Campylobacter coli strains from food animals.

Campylobacter coli is a food-borne pathogen associated increasingly with human gastroenteritis. C. coli has a high prevalence in swine, but is isolated also from cattle and poultry. Multilocus sequence typing (MLST) systems have been developed to differentiate C. coli strains. Although substantial allelic diversity was identified across all seven C. coli MLST loci, no correlations were made in two previous studies between allele or sequence type (ST) and the source of the organism. However, this may be due to either the relatively small number or the low diversity of C. coli strains used to validate both MLST studies. This study describes the typing of 488 C. coli strains from 4 different food animal sources (cattle, chickens, swine and turkeys), collected at different times over a 6 year period from different USA geographical locations. A total of 149 STs were identified. The 185 swine strains were the most diverse, possessing 82 STs. The cattle strains were the most clonal; 52/63 (83 %) strains possessed a single ST (ST-1068). A subpopulation of C. coli strains, collected primarily from turkeys, was identified, containing both C. coli- and Campylobacter jejuni-associated MLST alleles, specifically the C. jejuni allele aspA103. The majority of STs and alleles were host associated, i.e. found primarily in strains from a single food-animal source. Only 12/149 (8 %) STs were found in multiple sources. Additionally, the majority (34/46, 74 %) of major (n>5) alleles were more prevalent in certain hosts (swine, poultry). The presence of host-associated C. coli MLST alleles could lead potentially to more efficient source tracking in this species, especially in the trace-back of both sporadic and outbreak human clinical C. coli strains to animal sources.

Alleles↗

PubMed2006

Development of a multilocus sequence typing method for analysis of Lactobacillus plantarum strains.

Lactobacillus plantarum is a species of considerable industrial and medical interest. To date, the lack of reliable molecular methods for definite identification at strain level has hindered studies of the population biology of this organism. Here, a multilocus sequence typing (MLST) system for this organism is described, which exploits the genetic variation present in six housekeeping loci to determine the genetic relationship among isolates. The MLST system was established using 16 L. plantarum strains that were also characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S-23S rDNA intergenic spacer region (ISR). Ribotyping grouped the strains into four groups; however, RFLP analysis of the ISRs showed no differences in the strains analysed. In contrast, MLST had a good discriminatory ability. The sequence analysis of the six genes showed 14 different allelic combinations, with 12 of them represented by only one strain. By using this MLST approach we were able to confirm the identity of two strains deposited in the Spanish Type Culture Collection as different strains. Phylogenetic analysis indicated a panmictic population structure of L. plantarum and split decomposition analysis indicated that recombination plays a role in creating genetic heterogeneity in L. plantarum. As MLST allows precise identification, and easy comparison and exchange of results obtained in different laboratories, the future application of this new molecular method could be useful for the identification of valuable L. plantarum strains.

Bacterial Typing Techniques↗

PubMed2001

Multilocus sequence typing of Streptococcus pyogenes and the relationships between emm type and clone.

Multilocus sequence typing (MLST) is a tool that can be used to study the molecular epidemiology and population genetic structure of microorganisms. A MLST scheme was developed for Streptococcus pyogenes and the nucleotide sequences of internal fragments of seven selected housekeeping loci were obtained for 212 isolates. A total of 100 unique combinations of housekeeping alleles (allelic profiles) were identified. The MLST scheme was highly concordant with several other typing methods. The emm type, corresponding to a locus that is subject to host immune selection, was determined for each isolate; of the >150 distinct emm types identified to date, 78 are represented in this report. For a given emm type, the majority of isolates shared five or more of the seven housekeeping alleles. Stable associations between emm type and MLST were documented by comparing isolates obtained decades apart and/or from different continents. For the 33 emm types for which more than one isolate was examined, only five emm types were present on widely divergent backgrounds, differing at four or more of the housekeeping loci. The findings indicate that the majority of emm types examined define clones or clonal complexes. In addition, an MLST database is made accessible to investigators who seek to characterize other isolates of this species via the internet (http://www.mlst.net).

Alleles↗

PubMed2006

New Staphylococcus aureus genotyping method based on exotoxin (set) genes.

A variety of methods for genotyping Staphylococcus aureus isolates exists: the two most widely used methods are pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Here, we describe a sequence-based genotyping method based on genes encoding S. aureus superantigen-like proteins, which belong to a family of exotoxins called staphylococcal exotoxins. The sequences of PCR-amplified internal fragments of three different set genes (set2, set5, and set7) of 61 well-characterized clinical methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates and reference strains were compared. Phylogenetic analysis was performed based on single-nucleotide polymorphisms (SNP). The SNP dendrograms of the set gene sequences differentiated the 61 isolates into 22 distinct subgroups, designated exotoxin sequence types (ETST), while the standard seven-gene MLST profiles differentiated the same 61 isolates into 19 subgroups. Of the 19 different MLST subgroups, 16 corresponded to 16 distinct ETST groups. However, three MLST subgroups, ST1, ST30, and ST36, were each further separated into more than one ETST subgroup. The exotoxin-based genotyping method was able to discriminate MRSA and MSSA isolates according to their specific epidemiological characteristics. This SNP analysis of the three set genes is thus equally or more discriminatory than the seven-gene MLST method, providing a good alternative typing tool for a laboratory that has sequencing capability.

Bacterial Proteins↗

PubMed2006

Clonal complexes of Campylobacter jejuni identified by multilocus sequence typing are reliably predicted by restriction fragment length polymorphism analyses of the flaA gene.

Multilocus sequence typing (MLST) has provided important new insights into the population structure of Campylobacter jejuni and is rapidly becoming the gold standard for typing this species. However, the methodology is comparatively costly and slow to perform for the routine surveillance testing of large numbers of isolates required by public health laboratories. Restriction fragment length polymorphism analysis of the flaA gene (RFLP-flaA) and sequencing of the variable region in the fla locus (SVR-fla) were compared to MLST to determine if a low cost alternative could be found that reliably predicts clonal lineage (as determined by MLST). An isolate of C. jejuni from each of 153 patients from New South Wales, Australia, collected sequentially over a period of 30 months from 1999 to 2001 and comprising 40 sequence types (ST) from 15 clonal complexes (CC) was examined. Of 15 CC, 12 were represented by more than one isolate and a predominant RFLP-flaA type was found for 10 (83%). Of these, seven (70%) correctly predicted the predominant MLST CC with a probability of >0.8. Of 40 STs detected, 19 were reported for the first time, 9 of which were represented by more than one isolate. Eight of these were represented by a single RFLP-flaA type. Only two of eight major SVR-fla types were able to predict CC with a probability of >0.8, indicating that flaA-RFLP is a more reliable predictor of CC than SVR-fla and thus offers an alternative to MLST for use in routine surveillance.

Bacterial Typing Techniques↗

PubMed2006

Strain typing and determination of population structure of Candida krusei by multilocus sequence typing.

A multilocus sequence typing (MLST) scheme for Candida krusei was devised, based on sequencing of six gene fragments of the species. The existence of heterozygous results for each of the six fragments sequenced confirms that C. krusei is diploid for at least part of its genome. The C. krusei MLST scheme had a discriminatory index of 0.998, making this system ideal for strain typing of C. krusei clinical isolates. MLST data for 122 independent C. krusei isolates from a range of geographical sources were analyzed by eBURST, structure, and the unweighted-pair group method using average linkages to derive a population structure comprising four subtype strain clusters. There was no evidence of geographical associations with particular subtypes. Data for pairs of isolates from seven patients showed that each patient was colonized and/or infected with strain types that were indistinguishable by MLST. The C. krusei MLST database can be accessed online at http://pubmlst.org/ckrusei/.

Blood↗

PubMed2006

Multilocus sequence typing scheme for Enterococcus faecalis reveals hospital-adapted genetic complexes in a background of high rates of recombination.

A multilocus sequence typing (MLST) scheme based on seven housekeeping genes was used to investigate the epidemiology and population structure of Enterococcus faecalis. MLST of 110 isolates from different sources and geographic locations revealed 55 different sequence types that grouped into four major clonal complexes (CC2, CC9, CC10, and CC21) by use of eBURST. Two of these clonal complexes, CC2 and CC9, are particularly fit in the hospital environment, as CC2 includes the previously described BVE clonal complex identified by an alternative MLST scheme and CC9 includes exclusively isolates from hospitalized patients. Identical alleles were found in genetically diverse isolates with no linkage disequilibrium, while the different MLST loci gave incongruent phylogenetic trees. This demonstrates that recombination is an important mechanism driving genetic variation in E. faecalis and suggests an epidemic population structure for E. faecalis. Our novel MLST scheme provides an excellent tool for investigating local and short-term epidemiology as well as global epidemiology, population structure, and genetic evolution of E. faecalis.

Alleles↗

PubMed2002

Usefulness of multilocus sequence typing for characterization of clinical isolates of Candida albicans.

Molecular characterization of Candida albicans isolates is essential for understanding the epidemiology of nosocomial infections caused by this yeast. Here, we investigated the potential value of multilocus sequence typing (MLST) for characterizing epidemiologically related or unrelated C. albicans strains of various clinical origins. Accordingly, we sequenced the internal regions (loci) of six selected housekeeping genes of 40 C. albicans clinical isolates and 2 reference strains. In all, 68 polymorphic nucleotide sites were identified, of which 65 were found to be heterozygous in at least one isolate. Ten to 24 different genotypes were observed at the different loci, resulting, when combined, in 39 unique genotype combinations or diploid sequence types (DSTs). When MLST was applied to 26 epidemiologically unrelated isolates and the 2 reference strains, it allowed the identification of 27 independent DSTs, thus demonstrating a discriminatory power of 99.7. Using multidimensional scaling together with the minimum spanning tree method to analyze interstrain relationships, we identified six groups of genetically related isolates on the basis of bootstrap values of greater than 900. Application of MLST to 14 epidemiologically related isolates showed that those recovered from patients in the same hospital ward during the same 3 months had specific DSTs, although 73% of these isolates were genetically very close. This suggests that MLST can trace minute variations in the sequences of related isolates. Overall, MLST proved to be a highly discriminatory and stable method for unambiguous characterization of C. albicans.

Bacterial Proteins↗

PubMed2003

Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: strain diversity, host range, and recombination.

Three molecular typing methods were used to study the relationships among 184 Campylobacter strains isolated from humans, cattle, and chickens. All strains were genotyped by amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and sequence analysis of a genomic region with short tandem repeats designated clustered regularly interspaced short palindromic repeats (CRISPRs). MLST and AFLP analysis yielded more than 100 different profiles and patterns, respectively. These multiple-locus typing methods resulted in similar genetic clustering, indicating that both are useful in disclosing genetic relationships between Campylobacter jejuni isolates. Group separation analysis of the AFLP analysis and MLST data revealed an unexpected association between cattle and human strains, suggesting a common source of infection. Analysis of the polymorphic CRISPR region carrying short repeats allowed about two-thirds of the typeable strains to be distinguished, similar to AFLP analysis and MLST. The three methods proved to be equally powerful in identifying strains from outbreaks of human campylobacteriosis. Analysis of the MLST data showed that intra- and interspecies recombination occurs frequently and that the role of recombination in sequence variation is 50 times greater than that of mutation. Examination of strains cultured from cecum swabs revealed that individual chickens harbored multiple Campylobacter strain types and that some genotypes were found in more than one chicken. We conclude that typing of Campylobacter strains is useful for identification of outbreaks but is probably not useful for source tracing and global epidemiology because of carriage of strains of multiple types and an extremely high diversity of strains in animals.

Alleles↗

PubMed2003

Multilocus sequence typing of Staphylococcus aureus with DNA array technology.

A newly developed oligonucleotide array suited for multilocus sequence typing (MLST) of Staphylococcus aureus strains was analyzed with two strain collections in a two-center study. MLST allele identification for the first strain collection fully agreed with conventional strain typing. Analysis of strains from the second collection revealed that chip-defined MLST was concordant with conventional MLST. Array-mediated MLST data were reproducible, exchangeable, and epidemiologically concordant.

Alleles↗

PubMed2004

Comparative analysis of multilocus sequence typing and pulsed-field gel electrophoresis for characterizing Listeria monocytogenes strains isolated from environmental and clinical sources.

One hundred seventy-five Listeria monocytogenes strains were characterized by serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) based on loci in actA, betL, hlyA, gyrB, pgm, and recA. One hundred twenty-two sequence types (STs) were identified by MLST based on allelic profiles of the four housekeeping genes (betL, gyrB, pgm, and recA), and 34 and 38 alleles were identified for hlyA and actA, respectively. Several actA and hlyA alleles appeared to be predominantly associated with clinical isolates. MLST differentiated most of the L. monocytogenes strains better than did PFGE, and the discriminating ability of PFGE was better than that of serotyping. Several strains with different serotypes were found, by MLST and PFGE, to have very closely related genetic backgrounds, which suggested possible "antigen switching" among them. MLST can be a useful typing tool for differentiating L. monocytogenes strains (including strains undistinguishable by PFGE typing and serotyping), and it may be of value during investigations of food-borne outbreaks of listeriosis.

Bacterial Typing Techniques↗

PubMed2004

Multiple-locus variable-number tandem repeat analysis, a novel typing scheme to study the genetic relatedness and epidemiology of Enterococcus faecium isolates.

Multiresistant Enterococcus faecium is a major cause of hospital acquired infections and outbreaks. Here, we describe the development of multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) as a novel typing method to assess the genetic relatedness of E. faecium isolates. Six VNTR loci were used to genotype 392 isolates recovered from different animals and human community, hospital survey, and clinical isolates. From 3 to 13 alleles were found per locus, resulting in 127 different MLVA profiles. Clustering of MLVA profiles confirmed the host-specific genogroups found by multilocus sequence typing (MLST) and showed the grouping of clinical and epidemic isolates that belonged to the MLST-C1 cluster in a distinct MLVA-C1 cluster (sensitivity of 97% and specificity of 90%). Furthermore, the discriminatory power of MLVA is comparable to MLST. MLVA profiles appeared to be relatively stable, since isolates from a single outbreak shared the same MLVA profile, which is a prerequisite when MLVA is used to study hospital outbreaks. Our data show that MLVA is a highly reproducible and portable typing method; in contrast to MLST, it is fast, relatively cheap, and easy to perform. Furthermore, it has the abilities of MLST to recognize genetically related and potential epidemic isolates. Submission of MLVA profiles is possible via a Web-based database for international comparison.

Bacterial Typing Techniques↗

PubMed2005

Campylobacter coli in swine production: antimicrobial resistance mechanisms and molecular epidemiology.

The aim of this study was to determine antimicrobial resistance, to evaluate and compare the use of two genotyping methods for molecular epidemiology purposes, and to determine the genotypic diversity of Campylobacter coli of porcine origin. A total of 100 C. coli isolates from swine were tested for susceptibility to six antimicrobials using the agar dilution method and genotyped using two high-resolution fingerprinting approaches: multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Evaluation of the methods was based on their resistance patterns, discriminatory indexes (DI), high test throughputs, costs, and turnaround times. Resistance to erythromycin and tetracycline was the most common. Both genotypic methods were found to have high discriminatory power, although MLST had a higher DI (0.936) than PFGE (DI = 0.889). It also had a higher throughput than PFGE. Isolates were clustered into 27 groups by MLST compared to 11 by PFGE. MLST was able to further discriminate the isolates grouped under the same cluster by PFGE. Out of the 65 MLST sequence types (STs) identified among the total isolates, 50 were reported for the first time. Most STs were found to be specific to the farm (n = 38) and to slaughter (n = 22). Resistance against tetracycline and erythromycin was encoded by the tet(O) gene and a A2075G point mutation in the 23S rRNA gene, respectively. A high ciprofloxacin MIC (>64 microg/liter) was conferred by a point mutation in the gyrA gene. The weak clonal structure of the C. coli population among swine was further highlighted by the index of association value of 0.293. The findings of this study indicate that multidrug-resistant diverse C. coli strains exhibiting resistance to ciprofloxacin and erythromycin are concerning, since these are the drugs of choice for treating invasive campylobacteriosis cases in humans.

Abattoirs↗

PubMed2005

Multilocus sequence typing of Neisseria meningitidis directly from clinical samples and application of the method to the investigation of meningococcal disease case clusters.

Infections associated with Neisseria meningitidis are a major public health problem in England, Wales, and Northern Ireland. Currently, over 40% of cases are confirmed directly from clinical specimens using PCR-based methodologies without an organism being isolated. A nested/seminested multilocus sequence typing (MLST) system was developed at the Health Protection Agency Meningococcal Reference Unit to allow strain characterization beyond the serogroup for cases confirmed by PCR only. This system was evaluated on a panel of 20 meningococcus-positive clinical specimens (3 cerebrospinal fluid and 17 blood samples) from different patients containing various concentrations of meningococcal DNA that had corresponding N. meningitidis isolates. In each case, the sequence type generated from the clinical specimens matched that produced from the corresponding N. meningitidis isolate; the sensitivity of the MLST system was determined to be less than 12 genome copies per PCR. The MLST system was then applied to 15 PCR meningococcus-positive specimens (2 cerebrospinal fluid and 13 blood samples), each from a different patient, involved in three case clusters (two serogroup B and one serogroup W135) for which no corresponding N. meningitidis organisms had been isolated. In each case, an MLST sequence type was generated, allowing the accurate assignment of individual cases within each of the case clusters. In summary, the adaptation of the N. meningitidis MLST to a sensitive nested/seminested format for strain characterization directly from clinical specimens provides an important tool for surveillance and management of meningococcal infection.

Bacterial Proteins↗

PubMed2005

Use of variable-number tandem repeats to examine genetic diversity of Neisseria meningitidis.

Repetitive DNA motifs with potential variable-number tandem repeats (VNTR) were identified in the genome of Neisseria meningitidis and used to develop a typing method. A total of 146 meningococcal isolates recovered from carriers and patients were studied. These included 82 of the 107 N. meningitidis isolates previously used in the development of multilocus sequence typing (MLST), 45 isolates recovered from different counties in Norway in connection with local outbreaks, and 19 serogroup W135 isolates of sequence type 11 (ST-11), which were recovered in several parts of the world. The latter group comprised isolates related to the Hajj outbreak of 2000 and isolates recovered from outbreaks in Burkina Faso in 2001 and 2002. All isolates had been characterized previously by MLST or multilocus enzyme electrophoresis (MLEE). VNTR analysis showed that meningococcal isolates with similar MLST or MLEE types recovered from epidemiologically linked cases in a defined geographical area often presented similar VNTR patterns while isolates of the same MLST or MLEE types without an obvious epidemiological link showed variable VNTR patterns. Thus, VNTR analysis may be used for fine typing of meningococcal isolates after MLST or MLEE typing. The method might be especially valuable for differentiating among ST-11 strains, as shown by the VNTR analyses of serogroup W135 ST-11 meningococcal isolates recovered since the mid-1990s.

Bacterial Typing Techniques↗

PubMed2005

Multilocus sequence typing versus pulsed-field gel electrophoresis for characterization of extended-spectrum beta-lactamase-producing Escherichia coli isolates.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains are emerging pathogens. Molecular typing of ESBL-producing E. coli is useful for surveillance purposes, to monitor outbreaks and track nosocomial spread. Although pulsed-field gel electrophoresis (PFGE) is the current "gold standard" for bacterial molecular typing, multilocus sequence typing (MLST) may offer advantages. Forty ESBL-producing E. coli isolates were selected at random from a cohort of intensive care unit patients who had active surveillance perirectal cultures done. PFGE identified 19 unique PFGE types (PT) among the 40 isolates; MLST identified 22 unique sequence types. MLST had greater discriminatory ability than PFGE for ESBL-producing E. coli. Simpson's indices of diversity for PFGE and MLST were 0.895 and 0.956, respectively. There were five clonal complexes (CCs) (isolates with differences of no more than two loci) that each contained multiple PT, but each PT was found in only one CC, indicating genetic consistency within a CC. MLST has clear utility in studies of ESBL-producing E. coli, based on a greater discriminatory ability and reproducibility than PFGE and the ability to a priori define genetically related bacterial strains.

Bacterial Typing Techniques↗