scholarly journals Core Genome Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Anna Janowicz ◽  
Fabrizio De Massis ◽  
Massimo Ancora ◽  
Cesare Cammà ◽  
Claudio Patavino ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Multilocus Sequence Typing ◽  
Core Genome ◽  
Brucella Melitensis ◽  
Reference Sequence ◽  
Snp Analysis ◽  
Phylogenetic Distance ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type

ABSTRACT The use of whole-genome sequencing (WGS) using next-generation sequencing (NGS) technology has become a widely accepted method for microbiology laboratories in the application of molecular typing for outbreak tracing and genomic epidemiology. Several studies demonstrated the usefulness of WGS data analysis through single-nucleotide polymorphism (SNP) calling from a reference sequence analysis for Brucella melitensis, whereas gene-by-gene comparison through core-genome multilocus sequence typing (cgMLST) has not been explored so far. The current study developed an allele-based cgMLST method and compared its performance to that of the genome-wide SNP approach and the traditional multilocus variable-number tandem repeat analysis (MLVA) on a defined sample collection. The data set was comprised of 37 epidemiologically linked animal cases of brucellosis as well as 71 isolates with unknown epidemiological status, composed of human and animal samples collected in Italy. The cgMLST scheme generated in this study contained 2,704 targets of the B. melitensis 16M reference genome. We established the potential criteria necessary for inclusion of an isolate into a brucellosis outbreak cluster to be ≤6 loci in the cgMLST and ≤7 in WGS SNP analysis. Higher phylogenetic distance resolution was achieved with cgMLST and SNP analysis than with MLVA, particularly for strains belonging to the same lineage, thereby allowing diverse and unrelated genotypes to be identified with greater confidence. The application of a cgMLST scheme to the characterization of B. melitensis strains provided insights into the epidemiology of this pathogen, and it is a candidate to be a benchmark tool for outbreak investigations in human and animal brucellosis.

scholarly journals Core Genome Multi Locus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections

2018 ◽  
Author(s):  
Anna Janowicz ◽  
Fabrizio De Massis ◽  
Massimo Ancora ◽  
Cesare Cammà ◽  
Claudio Patavino ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Core Genome ◽  
Brucella Melitensis ◽  
Reference Sequence ◽  
Polymorphism Analysis ◽  
Snp Analysis ◽  
Phylogenetic Distance ◽  
Sample Collection ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

AbstractThe use of whole genome sequencing (WGS) using next generation sequencing (NGS) technology has become a widely accepted method for microbiology laboratories in the application of molecular typing for outbreak tracing and genomic epidemiology. Several studies demonstrated the usefulness of WGS data analysis through Single Nucleotide Polymorphism (SNP) calling from a reference sequence analysis for Brucella melitensis, whereas gene-by-gene comparison through core-genome Multilocus Sequence Typing (cgMLST) has not been explored so far. The current study developed an allele-based method cgMLST and compared its performance to the genome-wide SNP approach and the traditional MLVA on a defined sample collection. The dataset comprised of 37 epidemiologically linked animal cases of brucellosis as well as 71 epidemiologically unrelated human and animal isolates collected in Italy. The cgMLST scheme generated in this study contained 2,687 targets of the B. melitensis 16M reference genome (75.4% of the complete genome). We established the potential criteria necessary for inclusion of an isolate into a brucellosis outbreak cluster to be ≤4 loci in the cgMLST and ≤10 in WGS SNP analysis. CgMLST and SNP analysis provided much higher phylogenetic distance resolution than MLVA, particularly for strains belonging to the same lineage thus allowing diverse and unrelated genotypes to be identified with greater confidence. The application of this cgMLST scheme to the characterization of B. melitensis strains provided insights into the epidemiology of this pathogen and it is a candidate to be a benchmark tool for outbreak investigations in human and animal brucellosis.

scholarly journals Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

2014 ◽  
Vol 80 (7) ◽  
pp. 2125-2132 ◽  
Author(s):  
Narjol Gonzalez-Escalona ◽  
Ruth Timme ◽  
Brian H. Raphael ◽  
Donald Zink ◽  
Shashi K. Sharma
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Clostridium Botulinum ◽  
Toxin Gene ◽  
Polymorphism Analysis ◽  
Snp Analysis ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
Group I

ABSTRACTClostridium botulinumis a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA+OrfX−) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA−OrfX+) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producingC. botulinumstrains: two strains with the HA+OrfX−cluster (69A and 32A) and one strain with the HA−OrfX+cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly availableC. botulinumgroup I strains revealed five distinct lineages. Strains 69A and 32A clustered with theC. botulinumtype A1 Hall group, and strain CDC297 clustered with theC. botulinumtype Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination ofC. botulinumgroup I strains and demonstrates the utility of this analysis in quickly differentiatingC. botulinumstrains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.

scholarly journals Whole Genome and Core Genome Multilocus Sequence Typing and Single Nucleotide Polymorphism Analyses of Listeria monocytogenes Isolates Associated with an Outbreak Linked to Cheese, United States, 2013

2017 ◽  
Vol 83 (15) ◽  
Author(s):  
Yi Chen ◽  
Yan Luo ◽  
Heather Carleton ◽  
Ruth Timme ◽  
David Melka ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Clustering Analysis ◽  
Multilocus Sequence Typing ◽  
Core Genome ◽  
Variant Calling ◽  
Discriminatory Power ◽  
Sufficient Evidence ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

ABSTRACT Epidemiological findings of a listeriosis outbreak in 2013 implicated Hispanic-style cheese produced by company A, and pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) were performed on clinical isolates and representative isolates collected from company A cheese and environmental samples during the investigation. The results strengthened the evidence for cheese as the vehicle. Surveillance sampling and WGS 3 months later revealed that the equipment purchased by company B from company A yielded an environmental isolate highly similar to all outbreak isolates. The whole genome and core genome multilocus sequence typing and single nucleotide polymorphism (SNP) analyses results were compared to demonstrate the maximum discriminatory power obtained by using multiple analyses, which were needed to differentiate outbreak-associated isolates from a PFGE-indistinguishable isolate collected in a nonimplicated food source in 2012. This unrelated isolate differed from the outbreak isolates by only 7 to 14 SNPs, and as a result, the minimum spanning tree from the whole genome analyses and certain variant calling approach and phylogenetic algorithm for core genome-based analyses could not provide differentiation between unrelated isolates. Our data also suggest that SNP/allele counts should always be combined with WGS clustering analysis generated by phylogenetically meaningful algorithms on a sufficient number of isolates, and the SNP/allele threshold alone does not provide sufficient evidence to delineate an outbreak. The putative prophages were conserved across all the outbreak isolates. All outbreak isolates belonged to clonal complex 5 and serotype 1/2b and had an identical inlA sequence which did not have premature stop codons. IMPORTANCE In this outbreak, multiple analytical approaches were used for maximum discriminatory power. A PFGE-matched, epidemiologically unrelated isolate had high genetic similarity to the outbreak-associated isolates, with as few as 7 SNP differences. Therefore, the SNP/allele threshold should not be used as the only evidence to define the scope of an outbreak. It is critical that the SNP/allele counts be complemented by WGS clustering analysis generated by phylogenetically meaningful algorithms to distinguish outbreak-associated isolates from epidemiologically unrelated isolates. Careful selection of a variant calling approach and phylogenetic algorithm is critical for core-genome-based analyses. The whole-genome-based analyses were able to construct the highly resolved phylogeny needed to support the findings of the outbreak investigation. Ultimately, epidemiologic evidence and multiple WGS analyses should be combined to increase confidence levels during outbreak investigations.

scholarly journals Draft Genome Sequences of Two Natural Isolates of the Yeast Barnettozyma californica from Ireland, UCD09 and UCD89

2018 ◽  
Vol 6 (25) ◽  
Author(s):  
Alisha A. Mullen ◽  
Ciara D. Lynch ◽  
Shannon M. Hill ◽  
Cian P. Holohan ◽  
Tadhg Ó Cróinín ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Draft Genome ◽  
Snp Analysis ◽  
Nucleotide Polymorphism ◽  
Genome Sequences ◽  
Yeast Genus ◽  
Single Nucleotide ◽  
Content Type ◽  
The Family ◽  
Natural Isolates

ABSTRACT No genome sequence of a species from Barnettozyma, a yeast genus in the family Phaffomycetaceae, is currently available. We isolated two B. californica strains from soils in Ireland and generated draft sequences of their 11.7-Mb genomes. Single nucleotide polymorphism (SNP) analysis showed 20,490 differences between the strains and suggests that B. californica is haploid.

scholarly journals Differential Single Nucleotide Polymorphism-Based Analysis of an Outbreak Caused by Salmonella enterica Serovar Manhattan Reveals Epidemiological Details Missed by Standard Pulsed-Field Gel Electrophoresis

2015 ◽  
Vol 53 (4) ◽  
pp. 1227-1238 ◽  
Author(s):  
Erika Scaltriti ◽  
Davide Sassera ◽  
Francesco Comandatore ◽  
Marina Morganti ◽  
Carmen Mandalari ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Gel Electrophoresis ◽  
Salmonella Enterica ◽  
Pulsed Field Gel Electrophoresis ◽  
Snp Analysis ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
Pulsed Field ◽  
Codon Positions

We retrospectively analyzed a rareSalmonella entericaserovar Manhattan outbreak that occurred in Italy in 2009 to evaluate the potential of new genomic tools based on differential single nucleotide polymorphism (SNP) analysis in comparison with the gold standard genotyping method, pulsed-field gel electrophoresis. A total of 39 isolates were analyzed from patients (n= 15) and food, feed, animal, and environmental sources (n= 24), resulting in five different pulsed-field gel electrophoresis (PFGE) profiles. Isolates epidemiologically related to the outbreak clustered within the same pulsotype, SXB_BS.0003, without any further differentiation. Thirty-three isolates were considered for genomic analysis based on different sets of SNPs, core, synonymous, nonsynonymous, as well as SNPs in different codon positions, by Bayesian and maximum likelihood algorithms. Trees generated from core and nonsynonymous SNPs, as well as SNPs at the second and first plus second codon positions detailed four distinct groups of isolates within the outbreak pulsotype, discriminating outbreak-related isolates of human and food origins. Conversely, the trees derived from synonymous and third-codon-position SNPs clustered food and human isolates together, indicating that all outbreak-related isolates constituted a single clone, which was in line with the epidemiological evidence. Further experiments are in place to extend this approach within our regional enteropathogen surveillance system.

scholarly journals Molecular Epidemiology of Xanthomonas perforans Outbreaks in Tomato Plants from Transplant to Field as Determined by Single-Nucleotide Polymorphism Analysis

2019 ◽  
Vol 85 (18) ◽  
Author(s):  
Peter Abrahamian ◽  
Sujan Timilsina ◽  
Gerald V. Minsavage ◽  
Neha Potnis ◽  
Jeffrey B. Jones ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Genetic Variation ◽  
Snp Analysis ◽  
Bacterial Spot ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Tomato Plants ◽  
Content Type ◽  
Xanthomonas Perforans ◽  
A Genome

ABSTRACT Outbreaks of bacterial spot on tomato (BST) caused by Xanthomonas perforans are a major concern for sustainable crop production. BST is a common occurrence in tomato transplants grown for field production. We hypothesized that BST outbreaks in commercial fields originate from X. perforans strains inadvertently introduced from commercial transplant facilities. To test this hypothesis, we used a genome-wide single-nucleotide polymorphism (SNP) analysis to characterize X. perforans strains recovered from tomato transplant facilities and fields in commercial production areas. X. perforans strains were isolated from symptomatic transplants prior to roguing at two commercial transplant growers. Then, the same groups of transplants were tracked to commercial fields to recover X. perforans strains from diseased plants prior to harvest. Whole-genome sequencing was carried out on 84 strains isolated from transplant and field plants from Florida and South Carolina. SNPs were called using three reference strains that represented the genetic variation of the sampled strains. Field strains showing genetic similarity to transplant strains had a difference of 2 to 210 SNPs. Transplant and field strains clustered together by grower within each phylogenomic group, consistent with expectations. The range of genetic divergence among strains isolated from field plants was similar to the range obtained from strains on transplants. Using the range of genetic variation observed in transplants, we estimate that 60% to 100% of field strains were an extension of the transplant strain population. Our results stress the importance of BST management to reduce X. perforans movement from transplant to field and to minimize subsequent disease outbreaks. IMPORTANCE Current management of Xanthomonas perforans on tomato plants mainly relies on the frequent application of pesticides. However, the lack of effective pesticides and the development of strain tolerance to certain bactericides limit the ability to control outbreaks in production fields. Better knowledge of probable sources of X. perforans inoculum during tomato production is required to refine management strategies. Tomato plants are typically established in the field using transplants. This study aimed to determine if strains from field epidemics were coming from transplant facilities or resulted from local field outbreaks. The overall goal was to identify potential sources of inoculum and subsequently develop strategies to reduce carryover from transplant production to the field. Our results indicate that tomato producers should shift disease management efforts to transplant facilities to reduce disease in the field. Improved transplant health should reduce the likelihood of bacterial spot outbreaks and subsequently reduce pesticide usage in the field.

scholarly journals Genomic Investigation Reveals Contaminated Detergent as the Source of an Extended-Spectrum-β-Lactamase-Producing Klebsiella michiganensis Outbreak in a Neonatal Unit

2020 ◽  
Vol 58 (5) ◽  
Author(s):  
Paul Chapman ◽  
Brian M. Forde ◽  
Leah W. Roberts ◽  
Haakon Bergh ◽  
Debra Vesey ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Klebsiella Oxytoca ◽  
Multidrug Resistant ◽  
Hospital Environment ◽  
Nucleotide Polymorphism ◽  
Taxonomic Assignment ◽  
Single Nucleotide ◽  
Content Type ◽  
Extended Spectrum ◽  
Antimicrobial Resistance Genes

ABSTRACT Klebsiella species are problematic pathogens in neonatal units and may cause outbreaks, for which the sources of transmission may be challenging to elucidate. We describe the use of whole-genome sequencing (WGS) to investigate environmental sources of transmission during an outbreak of extended-spectrum-β-lactamase (ESBL)-producing Klebsiella michiganensis colonizing neonates. Ceftriaxone-resistant Klebsiella spp. isolated from neonates (or their mothers) and the hospital environment were included. Short-read sequencing (Illumina) and long-read sequencing (MinION; Oxford Nanopore Technologies) were used to confirm species taxonomy, to identify antimicrobial resistance genes, and to determine phylogenetic relationships using single-nucleotide polymorphism profiling. A total of 21 organisms (10 patient-derived isolates and 11 environmental isolates) were sequenced. Standard laboratory methods identified the outbreak strain as an ESBL-producing Klebsiella oxytoca, but taxonomic assignment from WGS data suggested closer identity to Klebsiella michiganensis. Strains isolated from multiple detergent-dispensing bottles were either identical or closely related by single-nucleotide polymorphism comparison. Detergent bottles contaminated by K. michiganensis had been used for washing milk expression equipment. No new cases were identified once the detergent bottles were removed. Environmental reservoirs may be an important source in outbreaks of multidrug-resistant organisms. WGS, in conjunction with traditional epidemiological investigation, can be instrumental in revealing routes of transmission and guiding infection control responses.

scholarly journals Leptin gene polymorphism of Ongole Grade cattle based on single nucleotide polymorphism

2018 ◽  
Vol 43 (4) ◽  
pp. 309
Author(s):  
N. Hilmia ◽  
D. Rahmat ◽  
D. Dudi
Keyword(s):  
Amino Acids ◽  
Single Nucleotide Polymorphism ◽  
Gene Polymorphism ◽  
Direct Sequencing ◽  
Snp Analysis ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Exon 2 ◽  
Specific Allele ◽  
Polymerase Chain

Point mutation on exon 2 of leptin gene, which changes amino acid encoding from Arginine to Cysteine, may alters the physiological function of the leptin hormone. This study aimed to identify leptin gene polymorphism of Ongole Grade (OG) cattle based on Single Nucleotide Polymorphism (SNP). The DNA sample was taken from 48 head of OG cattle at Balai Pengembangan Perbibitan Ternak Sapi Potong(BPPT SP) Cijeungjing West Java, which was isolated from white blood cell using the high salt method. Amplification of DNA was done by Polymerase Chain Reaction (PCR), followed by direct sequencing to obtain nucleotide sequence. The SNP analysis was carried out from alignment of sequencing result using Bioedit and MEGA 5.2 program. The results indicated in exon 2 leptin gene of OG cattle there was one synonymous SNPs that did not changeamino acids Serine encoding on g.1025T >C/S17S, while two non synonymous SNPaltered amino acids encoding, those were g.1047C> T /R25C and g.1048G>A/R25H. Those mutations changed amino acids encoding from Arginine to Cysteine and Arginine to Histidine respectively.In OG cattle, the frequency of A allele (44.8%) was higher than C allele (33.3%) and T allele (21.9%). Six genotypes were also identified, i.e. AA (41.7%), CC (20.8%), CT (20.8%), CA(4.2%), TT (10.4%) and TA (2.1 %). Heterozigosity of OG cattle based on leptin gene was 0.65 that was a high category. The A allele was a specific allele on Indonesian local cattle.

scholarly journals Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR34/L98H- and TR46/Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014

2015 ◽  
Vol 60 (1) ◽  
pp. 387-392 ◽  
Author(s):  
Faezeh Mohammadi ◽  
Seyed Jamal Hashemi ◽  
Jan Zoll ◽  
Willem J. G. Melchers ◽  
Haleh Rafati ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Quantitative Analysis ◽  
Single Nucleotide Polymorphisms ◽  
Aspergillus Fumigatus ◽  
Tandem Repeat ◽  
Promoter Region ◽  
Nucleotide Polymorphisms ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type

ABSTRACTWe employed an endpoint genotyping method to update the prevalence rate of positivity for the TR34/L98H mutation (a 34-bp tandem repeat mutation in the promoter region of thecyp51Agene in combination with a substitution at codon L98) and the TR46/Y121F/T289A mutation (a 46-bp tandem repeat mutation in the promoter region of thecyp51Agene in combination with substitutions at codons Y121 and T289) among clinicalAspergillus fumigatusisolates obtained from different regions of Iran over a recent 5-year period (2010 to 2014). The antifungal activities of itraconazole, voriconazole, and posaconazole against 172 clinicalA. fumigatusisolates were investigated using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. For the isolates with an azole resistance phenotype, thecyp51Agene and its promoter were amplified and sequenced. In addition, using a LightCycler 480 real-time PCR system, a novel endpoint genotyping analysis method targeting single-nucleotide polymorphisms was evaluated to detect the L98H and Y121F mutations in thecyp51Agene of all isolates. Of the 172A. fumigatusisolates tested, the MIC values of itraconazole (≥16 mg/liter) and voriconazole (>4 mg/liter) were high for 6 (3.5%). Quantitative analysis of single-nucleotide polymorphisms showed the TR34/L98H mutation in thecyp51Agenes of six isolates. No isolates harboring the TR46/Y121F/T289A mutation were detected. DNA sequencing of thecyp51Agene confirmed the results of the novel endpoint genotyping method. By microsatellite typing, all of the azole-resistant isolates had genotypes different from those previously recovered from Iran and from the Dutch TR34/L98H controls. In conclusion, there was not a significant increase in the prevalence of azole-resistantA. fumigatusisolates harboring the TR34/L98H resistance mechanism among isolates recovered over a recent 5-year period (2010 to 2014) in Iran. A quantitative assay detecting a single-nucleotide polymorphism in thecyp51Agene ofA. fumigatusis a reliable tool for the rapid screening and monitoring of TR34/L98H- and TR46/Y121F/T289A-positive isolates and can easily be incorporated into clinical mycology algorithms.

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