scholarly journals Variable-number tandem repeat markers for Mycobacterium intracellulare genotyping: comparison to the 16S rRNA gene sequencing

2017 ◽  
Vol 11 (02) ◽  
pp. 158-165 ◽  
Author(s):  
Kaisen Chen ◽  
Yangyi Zhang ◽  
Yiping Peng
Keyword(s):  
16S Rrna ◽  
Tandem Repeat ◽  
Variable Number Tandem Repeat ◽  
Point Mutations ◽  
Allelic Diversity ◽  
Variable Number ◽  
Clinical Samples ◽  
Group Method ◽  
Rrna Gene ◽  
Mycobacterium Intracellulare

Introduction: Characterizing Mycobacterium intracellulare responsible for nontuberculous mycobacterial (NTM) infections may aid in controlling outbreaks. This study aimed to compare 16S ribosomal ribonucleic acid (rRNA) sequencing and variable-number tandem repeat (VNTR) genotyping of M. intracellulare strains isolated from clinical samples, and to characterize VNTR clusters associated with NTM infections or cavity formation. Methodology: Sputum samples were obtained from 77 HIV-negative patients with pulmonary disease between 2009 and 2013. One M. intracellulare strain was isolated from each patient and genotyped using 16S rRNA and eight loci VNTR sequencing. Results: Single nucleotide polymorphism (SNP) genotyping identified seven point mutations at nucleotide positions 101, 178, 190, 252, 382, 443, and 490 in 16S rRNA, and four SNP patterns were identified: type 1 (16 strains), 2 (41 strains), 3 (11 strains), and 4 (1 strain); 5 strains had unique SNP patterns. VNTR genotyping identified VNTR12 as the most discriminating marker (allelic diversity 0.692). VNTR3 was the most homogeneous marker (allelic diversity 0.518), but each locus had high discriminating ability. The 77 strains were clustered according to the unpaired group method using arithmetic averages: cluster 1 (17 strains), 2 (43 strains), 3 (9 strains), and 4 (4 strains); 4strains had unique SNP patterns. Overall, over 90% strains were matched to similar SNP and VNTR groupings. VNTR clusters were associated with NTM infection (p =0.007) and presence of a cavity (p =0.042). Both methods distinguished four subtypes of M. intracellulare, which corresponded. Conclusions: VNTRs may represent an effective, user-friendly, low-cost typing technique.

scholarly journals Multiple-Locus Variable-Number Tandem Repeat Analysis Reveals Genetic Relationships within Bacillus anthracis

2000 ◽  
Vol 182 (10) ◽  
pp. 2928-2936 ◽  
Author(s):  
P. Keim ◽  
L. B. Price ◽  
A. M. Klevytska ◽  
K. L. Smith ◽  
J. M. Schupp ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Tandem Repeat ◽  
Genetic Relationships ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Group Method ◽  
Multiple Alleles ◽  
Multiple Locus ◽  
Sequence Characterization

ABSTRACT Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the B. anthracisgenome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers (vrrC 1, vrrC 2,vrrB 1, vrrB 2, and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously (vrrA). MLVA characterization of 426 B. anthracis isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.

scholarly journals High-Resolution In Situ Genotyping of Legionella pneumophila Populations in Drinking Water by Multiple-Locus Variable-Number Tandem-Repeat Analysis Using Environmental DNA

2010 ◽  
Vol 76 (18) ◽  
pp. 6186-6195 ◽  
Author(s):  
Leila Kahlisch ◽  
Karsten Henne ◽  
Josefin Draheim ◽  
Ingrid Brettar ◽  
Manfred G. Höfle
Keyword(s):  
Drinking Water ◽  
High Resolution ◽  
Tandem Repeat ◽  
Legionella Pneumophila ◽  
Variable Number Tandem Repeat ◽  
Environmental Dna ◽  
Variable Number ◽  
Small Scale ◽  
Rrna Gene ◽  
Multiple Locus

ABSTRACT Central to the understanding of infections by the waterborne pathogen Legionella pneumophila is its detection at the clonal level. Currently, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) of L. pneumophila isolates can be used as a tool for high-resolution genotyping. Since L. pneumophila is difficult to isolate, the isolation of outbreak strains often fails due to a viable but nonculturable (VBNC) state of the respective environmental population. Therefore, we developed a cultivation-independent approach to detect single clones in drinking water. This approach is based on the extraction of DNA from drinking water followed by PCR using a set of eight VNTR primer pairs necessary for MLVA genotyping of L. pneumophila. The PCR amplicons were analyzed by single-strand conformation polymorphism (SSCP) and capillary electrophoresis to obtain the respective MLVA profiles. Parallel to the high-resolution analysis, we used the same environmental DNA to quantify the number of L. pneumophila cells in drinking water using real-time PCR with 16S rRNA gene-targeted primers. We used a set of drinking water samples from a small-scale drinking water network to test our approach. With these samples we demonstrated that the developed approach was directly applicable to DNA obtained from drinking water. We were able to detect more L. pneumophila MLVA genotypes in drinking water than we could detect by isolation. Our approach could be a valuable tool to identify outbreak strains even after the outbreak has occurred and has the potential to be applied directly to clinical material.

scholarly journals Characterization of Novel Brucella Strains Originating from Wild Native Rodent Species in North Queensland, Australia

2010 ◽  
Vol 76 (17) ◽  
pp. 5837-5845 ◽  
Author(s):  
Rebekah V. Tiller ◽  
Jay E. Gee ◽  
Michael A. Frace ◽  
Trevor K. Taylor ◽  
Joao C. Setubal ◽  
...  
Keyword(s):  
16S Rrna ◽  
Variable Number Tandem Repeat ◽  
Housekeeping Genes ◽  
Variable Number ◽  
Rodent Species ◽  
Whole Genome Sequence ◽  
Rrna Gene ◽  
Microbiological Test ◽  
Group Of Seven

ABSTRACT We report on the characterization of a group of seven novel Brucella strains isolated in 1964 from three native rodent species in North Queensland, Australia, during a survey of wild animals. The strains were initially reported to be Brucella suis biovar 3 on the basis of microbiological test results. Our results indicated that the rodent strains had microbiological traits distinct from those of B. suis biovar 3 and all other Brucella spp. To reinvestigate these rodent strains, we sequenced the 16S rRNA, recA, and rpoB genes and nine housekeeping genes and also performed multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA). The rodent strains have a unique 16S rRNA gene sequence compared to the sequences of the classical Brucella spp. Sequence analysis of the recA, rpoB, and nine housekeeping genes reveals that the rodent strains are genetically identical to each other at these loci and divergent from any of the currently described Brucella sequence types. However, all seven of the rodent strains do exhibit distinctive allelic MLVA profiles, although none demonstrated an amplicon for VNTR 07, whereas the other Brucella spp. did. Phylogenetic analysis of the MLVA data reveals that the rodent strains form a distinct clade separate from the classical Brucella spp. Furthermore, whole-genome sequence comparison using the maximal unique exact matches index (MUMi) demonstrated a high degree of relatedness of one of the seven rodent Brucella strains (strain NF 2653) to another Australian rodent Brucella strain (strain 83-13). Our findings strongly suggest that this group of Brucella strains isolated from wild Australian rodents defines a new species in the Brucella genus.

scholarly journals Current advances in molecular subtyping using multilocus variable number of tandem repeat analysis of Salmonella Enteritidis and Salmonella Typhimurium in Egyptian chickens

2020 ◽  
Vol 13 (10) ◽  
pp. 2252-2259
Author(s):  
Wafaa M. M. Hassan ◽  
Ashraf A. Abd El Tawab ◽  
Sara M. El-Shannat
Keyword(s):  
Salmonella Typhimurium ◽  
Tandem Repeat ◽  
Disease Transmission ◽  
Salmonella Enteritidis ◽  
Profile Analysis ◽  
Variable Number Tandem Repeat ◽  
Allelic Diversity ◽  
Variable Number ◽  
Valuable Insight ◽  
Repeat Analysis

Aim: This study aimed to characterize the genetic diversity, evolutionary level, and prevalence of genotypes of common isolates of Salmonella (Salmonella Enteritidis and Salmonella Typhimurium). Using one of the most advanced molecular recognition techniques, multilocus variable number of tandem repeat analysis (MLVA), we characterized the genotype and prevalence of S. Enteritidis and S. Typhimurium. Materials and Methods: One hundred and twenty-five internal organ samples were collected from the major chicken slaughterhouses in Egypt, and Salmonella species were isolated. PCR was utilized to amplify the IE-1 and Flic-C genes to identify S. Enteritidis and S. Typhimurium DNA, respectively, from Salmonella isolates. MLVA was applied on nine samples of S. Enteritidis DNA and three samples of S. Typhimurium DNA. Six variable number tandem repeat (VNTR) loci (Sal02, Sal04, Sal06, Sal10, Sal20, and Sal23) were amplified. Results: Of the examined samples (n=125), a total of 12 isolates (9.6%) were either identified as Enteritidis or Typhimurium. PCR-mediated amplification of IE-1 and Flic-C revealed that 75% (n=9) of the 12 Salmonella isolates were S. Enteritidis and 25% (n=3) were S. Typhimurium. The six loci amplified through MLVA had allelic diversity. The most discriminatory heterogenic locus for S. Enteritidis was Sal20. Sal04 and Sal23 were the most discriminatory heterogenic loci for S. Typhimurium. VNTR allelic profile analysis revealed nine unique genotypes for S. Enteritidis and three for S. Typhimurium. Conclusion: This study was the first to use MLVA analysis to identify S. Enteritidis and S. Typhimurium strains isolated from chickens in Egypt. The molecular typing data reported herein allowed us to characterize the genotypes of S. Enteritidis and S. Typhimurium that are most prevalent in Egyptian chickens. Moreover, this epidemiological information provides valuable insight on how to prevent disease transmission. Moreover, our methods provide an alternative to traditional serotyping techniques that may produce inaccurate strain identifications for organisms with rough lipopolysaccharide structures.

scholarly journals Molecular Characterization of Korean Bacillus anthracis Isolates by Amplified Fragment Length Polymorphism Analysis and Multilocus Variable-Number Tandem Repeat Analysis

2005 ◽  
Vol 71 (8) ◽  
pp. 4664-4671 ◽  
Author(s):  
Chunsun Ryu ◽  
Kyunghee Lee ◽  
Han-Jun Hawng ◽  
Cheon-Kwon Yoo ◽  
Won-Keun Seong ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Length Polymorphism ◽  
Tandem Repeat ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Variable Number Tandem Repeat ◽  
Amplified Fragment Length Polymorphism ◽  
Variable Number ◽  
Clinical Samples ◽  
Polymorphism Analysis

ABSTRACT We analyzed the genetic relationships and molecular characteristics of 34 Bacillus anthracis isolates from soil and clinical samples in various regions of Korea and 17 related Bacillus species, using the amplified fragment length polymorphism (AFLP) and multilocus variable-number tandem repeat (MLVA) approaches. Triplicate AFLP profiles of these strains showed high reproducibility and identified 376 polymorphisms. AFLP phylogenetic analysis of B. anthracis isolates showed a high level of similarity, 0.93, and this monomorphic fragment profile proved to be useful to differentiate B. anthracis strains from other Bacillus species. The B. cereus group was separated from other Bacillus species at a level of similarity of 0.68. Among them, some B. cereus strains showed genetic interspersion with B. thuringiensis strains. The evolutionary pattern of nucleotide differences among B. anthracis strains with the eight MLVA markers showed nine MLVA types. Three MLVA types, M1 to M3, were pathogenic B. anthracis isolates and were assigned as new genotypes belonging to the A4 and B3 clusters, compared with 89 genotypes deduced from previous data. This indicates that differences in cluster prevalence and distribution may be influenced more by MLVA markers on two plasmids loci and human activity. Consequently, we suggest that the novel MLVA type may represent significant evidence for historic adaptation to environmental conditions of the Asian continent, particularly Korea. Therefore, MLVA techniques may be available for molecular monitoring on anthrax-release-related bioterrorism and further study is required for the continuous epidemiological study of variable anthrax collections.

scholarly journals Genetic diversity ofMycobacterium aviumcomplex strains isolated in Argentina by MIRU-VNTR

2017 ◽  
Vol 145 (7) ◽  
pp. 1382-1391 ◽  
Author(s):  
B. R. IMPERIALE ◽  
R. D. MOYANO ◽  
A. B. DI GIULIO ◽  
M. A. ROMERO ◽  
M. F. ALVARADO PINEDO ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Mycobacterium Avium ◽  
Tandem Repeat ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Geographical Region ◽  
Clinical Samples ◽  
Discriminatory Index

SUMMARYMycobacterium aviumsp.avium(MAA),M. aviumsp.hominissuis(MAH), andM. aviumsp.paratuberculosis(MAP) are the main members of theM. aviumcomplex (MAC) causing diseases in several hosts. The aim of this study was to describe the genetic diversity of MAC isolated from different hosts. Twenty-six MAH and 61 MAP isolates were recovered from humans and cattle, respectively. GenoType CM®and IS1311-PCR were used to identifyMycobacteriumspecies. The IS901-PCR was used to differentiate between MAH and MAA, while IS900-PCR was used to identify MAP. Genotyping was performed using a mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) scheme (loci: 292, X3, 25, 47, 3, 7, 10, 32) and patterns (INMV) were assigned according to the MAC-INMV database (http://mac-inmv.tours.inra.fr/). Twenty-two (22/26, 84·6%) MAH isolates were genotyped and 16 were grouped into the following, INMV 92, INMV 121, INMV 97, INMV 103, INMV 50, and INMV 40. The loci X3 and 25 showed the largest diversity (D: 0·5844), and the global discriminatory index (Hunter and Gaston discriminatory index, HGDI) was 0·9300. MAP (100%) isolates were grouped into INMV 1, INMV 2, INMV 11, INMV 8, and INMV 5. The HGDI was 0·6984 and loci 292 and 7 had the largestD(0·6980 and 0·5050). MAH presented a higherDwhen compared with MAP. The MIRU-VNTR was a useful tool to describe the genetic diversity of both MAH and MAP as well as to identify six new MAH patterns that were conveniently reported to the MAC-INMV database. It was also demonstrated that, in the geographical region studied, human MAC cases were produced by MAH as there was no MAA found among the human clinical samples.

scholarly journals Serological Survey and Molecular Typing Reveal New Leptospira Serogroup Pomona Strains among Pigs of Northern Italy

Pathogens ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 332 ◽  
Author(s):  
Cristina Bertasio ◽  
Alice Papetti ◽  
Erika Scaltriti ◽  
Silvia Tagliabue ◽  
Mario D’Incau ◽  
...  
Keyword(s):  
Serological Test ◽  
Variable Number Tandem Repeat ◽  
16S Rrna Gene Sequencing ◽  
Variable Number ◽  
Northern Italy ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Accurate Identification ◽  
Pathogenic Leptospira ◽  
Rrna Gene Sequencing

Swine act as both maintenance and incidental hosts of pathogenic Leptospira spp. Here, a serological test was performed on 131,660 pig sera collected between 2002 and 2017 from 4715 farms in Northern Italy. A positivity rate of 13.05% was determined. Australis was the most frequently identified serogroup (77.29%), followed by Pomona (18.47%), Tarassovi (1.51%) and Icterohaemorrhagie (1.40%). Culture isolation and real-time Polymerase chain reaction (PCR) were carried out on 347 kidneys and 470 clinical samples, respectively. Overall, 133 strains were cultured successfully and 43 randomly chosen isolates were identified as serogroup Pomona. Multi-locus sequence typing (MLST) revealed that 41 isolates and 8 DNA extracted from biological samples belonged to sequence type 140. Using a multiple-locus, variable-number tandem repeat analysis, 43 samples produced identical profiles but, after 2014, three new Leptospira interrogans serogroup Pomona genotypes were observed. Interestingly, two isolates showed new MLST profiles and an unclassified identification by monoclonal antibodies. The 16S rRNA gene sequencing clustered them into L. kirschneri species and a core genome MLST analysis revealed an allelic identity of 96% compared with Mozdok strains. Genotyping allowed us to discriminate leptospires and to identify new emerging strains. The accurate identification of infective strains is required for formulating preventive methods and intervention strategies.

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