scholarly journals A Naturally Occurring Single Nucleotide Polymorphism in a Multicopy Plasmid Produces a Reversible Increase in Antibiotic Resistance

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Alfonso Santos-Lopez ◽  
Cristina Bernabe-Balas ◽  
Manuel Ares-Arroyo ◽  
Rafael Ortega-Huedo ◽  
Andreas Hoefer ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Antibiotic Resistance ◽  
Experimental Evolution ◽  
Pasteurella Multocida ◽  
Single Mutation ◽  
Multicopy Plasmid ◽  
Nucleotide Polymorphism ◽  
Resistance Level ◽  
Single Nucleotide ◽  
Content Type

ABSTRACT ColE1 plasmids are small mobilizable replicons that play an important role in the spread of antibiotic resistance in Pasteurellaceae. In this study, we describe how a natural single nucleotide polymorphism (SNP) near the origin of replication of the ColE1-type plasmid pB1000 found in a Pasteurella multocida clinical isolate generates two independent plasmid variants able to coexist in the same cell simultaneously. Using the Haemophilus influenzae Rd KW20 strain as a model system, we combined antibiotic susceptibility tests, quantitative PCRs, competition assays, and experimental evolution to characterize the consequences of the coexistence of the pB1000 plasmid variants. This coexistence produced an increase of the total plasmid copy number (PCN) in the host bacteria, leading to a rise in both the antibiotic resistance level and the metabolic burden produced by pB1000. Using experimental evolution, we showed that in the presence of ampicillin, the bacteria maintained both plasmid variants for 300 generations. In the absence of antibiotics, on the other hand, the bacteria are capable of reverting to the single-plasmid genotype via the loss of one of the plasmid variants. Our results revealed how a single mutation in plasmid pB1000 provides the bacterial host with a mechanism to increase the PCN and, consequently, the ampicillin resistance level. Crucially, this mechanism can be rapidly reversed to avoid the extra cost entailed by the increased PCN in the absence of antibiotics.

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 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 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.

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 Updated Reference Genome Sequence and Annotation of Mycobacterium bovis AF2122/97

2017 ◽  
Vol 5 (14) ◽  
Author(s):  
Kerri M. Malone ◽  
Damien Farrell ◽  
Tod P. Stuber ◽  
Olga T. Schubert ◽  
Ruedi Aebersold ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Mycobacterium Bovis ◽  
Genome Sequence ◽  
Bovine Tuberculosis ◽  
Reference Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Coding Sequences ◽  
Content Type ◽  
Reference Genome Sequence

ABSTRACT We report here an update to the reference genome sequence of the bovine tuberculosis bacillus Mycobacterium bovis AF2122/97, generated using an integrative multiomics approach. The update includes 42 new coding sequences (CDSs), 14 modified annotations, 26 single-nucleotide polymorphism (SNP) corrections, and disclosure that the RD900 locus, previously described as absent from the genome, is in fact present.

scholarly journals A Whole-Genome Single Nucleotide Polymorphism-Based Approach To Trace and Identify Outbreaks Linked to a Common Salmonella enterica subsp. enterica Serovar Montevideo Pulsed-Field Gel Electrophoresis Type

2011 ◽  
Vol 77 (24) ◽  
pp. 8648-8655 ◽  
Author(s):  
Henk C. den Bakker ◽  
Andrea I. Moreno Switt ◽  
Craig A. Cummings ◽  
Karin Hoelzer ◽  
Lovorka Degoricija ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Gel Electrophoresis ◽  
Salmonella Enterica ◽  
Pfge Pattern ◽  
Pulsed Field Gel Electrophoresis ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
Pulsed Field

ABSTRACTIn this study, we report a whole-genome single nucleotide polymorphism (SNP)-based evolutionary approach to study the epidemiology of a multistate outbreak ofSalmonella entericasubsp.entericaserovar Montevideo. This outbreak included 272 cases that occurred in 44 states between July 2009 and April 2010. A case-control study linked the consumption of salami made with contaminated black and red pepper to the outbreak. We sequenced, on the SOLiD System, 47 isolates with XbaI PFGE pattern JIXX01.0011, a common pulsed-field gel electrophoresis (PFGE) pattern associated with isolates from the outbreak. These isolates represented 20 isolates collected from human sources during the period of the outbreak and 27 control isolates collected from human, food, animal, and environmental sources before the outbreak. Based on 253 high-confidence SNPs, we were able to reconstruct a tip-dated molecular clock phylogeny of the isolates and to assign four human isolates to the actual outbreak. We developed an SNP typing assay to rapidly discriminate between outbreak-related cases and non-outbreak-related cases and tested this assay on an extended panel of 112 isolates. These results suggest that only a very small percentage of the human isolates with the outbreak PFGE pattern and obtained during the outbreak period could be attributed to the actual pepper-related outbreak (20%), while the majority (80%) of the putative cases represented background cases. This study demonstrates that next-generation-based SNP typing provides the resolution and accuracy needed for outbreak investigations of food-borne pathogens that cannot be distinguished by currently used subtyping methods.

scholarly journals Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Iain C. Clark ◽  
Ryan A. Melnyk ◽  
Matthew D. Youngblut ◽  
Hans K. Carlson ◽  
Anthony T. Iavarone ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Copy Number ◽  
Shewanella Oneidensis ◽  
Respiratory Metabolism ◽  
Chromosome 11 ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Single Strain ◽  
Content Type ◽  
Essential Components

ABSTRACTDespite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction fromShewanella algaeACDC in the non-chlorate-reducingShewanella oneidensisMR-1. The construct that ultimately endowed robust growth on chlorate includedcld, a cytochromecgene,clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream ofcldenabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence betweennarQPandnrfAthat enhanced growth.IMPORTANCEThe ability of chlorate reduction composite transposons to form functional metabolisms after transfer to a new host is an important part of their propagation. To study this phenomenon, we engineeredShewanella oneidensisMR-1 into a chlorate reducer. We defined a set of genes sufficient to endow growth on chlorate from a plasmid, but found that chromosomal insertion of these genes was nonfunctional. Evolution of this inoperative strain into a chlorate reducer showed that tandem duplication was a dominant mechanism of activation. While copy number changes are a relatively rapid way of increasing gene dosage, replicating almost 1 megabase of extra DNA is costly. Mutations that alleviate the need for high copy number are expected to arise and eventually predominate, and we identified a single nucleotide polymorphism (SNP) that relieved the copy number requirement. This study uses both rational and evolutionary approaches to gain insight into the evolution of a fascinating respiratory metabolism.

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 Role of Interleukin-23 (IL-23) Receptor Signaling for IL-17 Responses in Human Lyme Disease

2011 ◽  
Vol 79 (11) ◽  
pp. 4681-4687 ◽  
Author(s):  
Marije Oosting ◽  
Hadewych ter Hofstede ◽  
Frank L. van de Veerdonk ◽  
Patrick Sturm ◽  
Bart-Jan Kullberg ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Lyme Disease ◽  
Gene Polymorphism ◽  
Taqman Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
Interleukin 23

ABSTRACTInterleukin-23 (IL-23) is known to play a crucial role in the development and maintenance of T helper 17 cells. It has been previously demonstrated that IL-17 is involved in experimental Lyme arthritis, caused byBorrelia burgdorferibacteria. However, the precise role of the IL-23 receptor (IL-23R) for theB. burgdorferi-induced IL-17 responses or human Lyme disease has not yet been elucidated. IL-23R single nucleotide polymorphism (SNP) rs11209026 was genotyped using the TaqMan assay. Functional studies were performed using peripheral blood mononuclear cells, and cytokines were measured using enzyme-linked immunosorbent assay (ELISA). Dose-dependent production of IL-23 and IL-17 byB. burgdorfericould be observed. Interestingly, when IL-23 bioactivity was inhibited by a specific antibody against IL-23p19, IL-17 production was significantly downregulated. In contrast, production of gamma interferon (IFN-γ) was not affected after the blockade of IL-23 activity. Moreover, individuals bearing a single nucleotide polymorphism in the IL-23R gene (Arg381Gln) produced significantly less IL-17 afterB. burgdorferistimulation compared with that of the individuals bearing the wild type. Despite lower IL-17 production, the IL-23R gene polymorphism did not influence the development of chronic Lyme disease in a cohort of patients with Lyme disease. This study demonstrates that IL-23R signaling is needed forB. burgdorferi-induced IL-17 productionin vitroand that an IL-23R gene SNP leads to impaired IL-17 production. However, the IL-23R gene polymorphism is not crucial for the pathogenesis of chronic Lyme.

scholarly journals Significance of Staphylococcus epidermidis in Health Care-Associated Infections, from Contaminant to Clinically Relevant Pathogen: This Is a Wake-Up Call!

2016 ◽  
Vol 54 (7) ◽  
pp. 1679-1681 ◽  
Author(s):  
Micael Widerström
Keyword(s):  
Health Care ◽  
Single Nucleotide Polymorphism ◽  
Staphylococcus Epidermidis ◽  
Clinical Microbiology ◽  
Nucleotide Polymorphism ◽  
Diagnostic Challenge ◽  
Coagulase Negative Staphylococci ◽  
Single Nucleotide ◽  
Content Type ◽  
Health Care Associated Infections

Coagulase-negative staphylococci, particularlyStaphylococcus epidermidis, have been recognized as an important cause of health care-associated infections. Concurrently,S. epidermidisis a common contaminant in clinical cultures, which poses a diagnostic challenge. An article in this issue ofJournal of Clinical Microbiology(I. Tolo, J. C. Thomas, R. S. B. Fischer, E. L. Brown, B. M. Gray, and D. A. Robinson, J Clin Microbiol 54:1711–1719, 2015,http://dx.doi.org/10.1128/JCM.03345-15) describes a rapid single nucleotide polymorphism-based assay for distinguishing betweenS. epidermidisisolates from hospital and nonhospital sources, which represents an important contribution to the characterization and understanding ofS. epidermidishealth care-associated infections.

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