scholarly journals Establishment and evaluation of a core genome multilocus sequence typing scheme for whole-genome sequence-based typing of Pseudomonas aeruginosa

2020 ◽  
pp. JCM.01987-20
Author(s):  
Hauke Tönnies ◽  
Karola Prior ◽  
Dag Harmsen ◽  
Alexander Mellmann
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multilocus Sequence Typing ◽  
Core Genome ◽  
Target Genes ◽  
Multidrug Resistant ◽  
Population Based ◽  
Whole Genome Sequence ◽  
Nucleotide Polymorphisms ◽  
Environmental Bacterium ◽  
Random Dataset

The environmental bacterium Pseudomonas aeruginosa, in particular multidrug resistant clones, is often associated with nosocomial infections and outbreaks. Today, core genome multilocus sequence typing (cgMLST) is frequently applied to delineate sporadic cases from nosocomial transmissions. However, until recently, no cgMLST scheme for a standardized typing of P. aeruginosa was available.To establish a novel cgMLST scheme for P. aeruginosa, we initially determined the breadth of the P. aeruginosa population based on MLST data with a Bayesian approach (BAPS). Using genomic data of representative isolates for the whole population and for all 12 serogroups, we extracted target genes and further refined them using a random dataset of 1,000 P. aeruginosa genomes. Subsequently, we investigated reproducibility and discriminatory ability with repeatedly sequenced isolates and isolates from well-defined outbreak scenarios, respectively, and compared clustering applying two recently published cgMLST schemes.BAPS generated seven P. aeruginosa groups. To cover these and all serogroups, 15 reference strains were used to determine genes common in all strains. After refinement with the dataset of 1,000 genomes, the cgMLST scheme consisted of 3,867 target genes, which are representative for the P. aeruginosa population and highly reproducible using biological replicates. We finally evaluated the scheme by reanalyzing two published outbreaks, where the authors used single nucleotide polymorphisms (SNPs) typing. In both cases cgMLST was concordant to the previous SNP results and to the results of the two other cgMLST schemes.In conclusion, the highly-reproducible novel P. aeruginosa cgMLST scheme facilitates outbreak investigations due to the publicly available cgMLST nomenclature.

scholarly journals Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Genome-Wide Typing of Clostridium difficile

2018 ◽  
Vol 56 (6) ◽  
Author(s):  
Stefan Bletz ◽  
Sandra Janezic ◽  
Dag Harmsen ◽  
Maja Rupnik ◽  
Alexander Mellmann
Keyword(s):  
Clostridium Difficile ◽  
Multilocus Sequence Typing ◽  
Core Genome ◽  
Target Genes ◽  
Population Based ◽  
Whole Genome Sequencing Data ◽  
The Novel ◽  
Sequencing Data ◽  
Gastrointestinal Infections ◽  
Content Type

ABSTRACT Clostridium difficile , recently renamed Clostridioides difficile , is the most common cause of antibiotic-associated nosocomial gastrointestinal infections worldwide. To differentiate endogenous infections and transmission events, highly discriminatory subtyping is necessary. Today, methods based on whole-genome sequencing data are increasingly used to subtype bacterial pathogens; however, frequently a standardized methodology and typing nomenclature are missing. Here we report a core genome multilocus sequence typing (cgMLST) approach developed for C. difficile . Initially, we determined the breadth of the C. difficile population based on all available MLST sequence types with Bayesian inference (BAPS). The resulting BAPS partitions were used in combination with C. difficile clade information to select representative isolates that were subsequently used to define cgMLST target genes. Finally, we evaluated the novel cgMLST scheme with genomes from 3,025 isolates. BAPS grouping ( n = 6 groups) together with the clade information led to a total of 11 representative isolates that were included for cgMLST definition and resulted in 2,270 cgMLST genes that were present in all isolates. Overall, 2,184 to 2,268 cgMLST targets were detected in the genome sequences of 70 outbreak-associated and reference strains, and on average 99.3% cgMLST targets (1,116 to 2,270 targets) were present in 2,954 genomes downloaded from the NCBI database, underlining the representativeness of the cgMLST scheme. Moreover, reanalyzing different cluster scenarios with cgMLST were concordant to published single nucleotide variant analyses. In conclusion, the novel cgMLST is representative for the whole C. difficile population, is highly discriminatory in outbreak situations, and provides a unique nomenclature facilitating interlaboratory exchange.

scholarly journals Core Genome Multilocus Sequence Typing Scheme for High-Resolution Typing of Enterococcus faecium

2015 ◽  
Vol 53 (12) ◽  
pp. 3788-3797 ◽  
Author(s):  
Mark de Been ◽  
Mette Pinholt ◽  
Janetta Top ◽  
Stefan Bletz ◽  
Alexander Mellmann ◽  
...  
Keyword(s):  
High Resolution ◽  
Enterococcus Faecium ◽  
Multilocus Sequence Typing ◽  
Core Genome ◽  
Target Genes ◽  
Multidrug Resistant ◽  
Single Nucleotide ◽  
Content Type ◽  
Genome Wide ◽  
Computationally Intensive

Enterococcus faecium, a common inhabitant of the human gut, has emerged in the last 2 decades as an important multidrug-resistant nosocomial pathogen. Since the start of the 21st century, multilocus sequence typing (MLST) has been used to study the molecular epidemiology ofE. faecium. However, due to the use of a small number of genes, the resolution of MLST is limited. Whole-genome sequencing (WGS) now allows for high-resolution tracing of outbreaks, but current WGS-based approaches lack standardization, rendering them less suitable for interlaboratory prospective surveillance. To overcome this limitation, we developed a core genome MLST (cgMLST) scheme forE. faecium. cgMLST transfers genome-wide single nucleotide polymorphism (SNP) diversity into a standardized and portable allele numbering system that is far less computationally intensive than SNP-based analysis of WGS data. TheE. faeciumcgMLST scheme was built using 40 genome sequences that represented the diversity of the species. The scheme consists of 1,423 cgMLST target genes. To test the performance of the scheme, we performed WGS analysis of 103 outbreak isolates from five different hospitals in the Netherlands, Denmark, and Germany. The cgMLST scheme performed well in distinguishing between epidemiologically related and unrelated isolates, even between those that had the same sequence type (ST), which denotes the higher discriminatory power of this cgMLST scheme over that of conventional MLST. We also show that in terms of resolution, the performance of theE. faeciumcgMLST scheme is equivalent to that of an SNP-based approach. In conclusion, the cgMLST scheme developed in this study facilitates rapid, standardized, and high-resolution tracing ofE. faeciumoutbreaks.

scholarly journals The Widespread Multidrug-Resistant Serotype O12 Pseudomonas aeruginosa Clone Emerged through Concomitant Horizontal Transfer of Serotype Antigen and Antibiotic Resistance Gene Clusters

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Sandra Wingaard Thrane ◽  
Véronique L. Taylor ◽  
Luca Freschi ◽  
Irena Kukavica-Ibrulj ◽  
Brian Boyle ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Horizontal Transfer ◽  
Core Genome ◽  
Gene Clusters ◽  
Multidrug Resistant ◽  
Clinical Settings ◽  
Content Type ◽  
The Past ◽  
Antibiotic Resistance Determinant

ABSTRACTThe O-specific antigen (OSA) inPseudomonas aeruginosalipopolysaccharide is highly varied by sugar identity, side chains, and bond between O-repeats. These differences classifiedP. aeruginosainto 20 distinct serotypes. In the past few decades, O12 has emerged as the predominant serotype in clinical settings and outbreaks. These serotype O12 isolates exhibit high levels of resistance to various classes of antibiotics. Here, we explore how theP. aeruginosaOSA biosynthesis gene clusters evolve in the population by investigating the association between the phylogenetic relationships among 83P. aeruginosastrains and their serotypes. While most serotypes were closely linked to the core genome phylogeny, we observed horizontal exchange of OSA biosynthesis genes among phylogenetically distinctP. aeruginosastrains. Specifically, we identified a “serotype island” ranging from 62 kb to 185 kb containing theP. aeruginosaO12 OSA gene cluster, an antibiotic resistance determinant (gyrAC248T), and other genes that have been transferred betweenP. aeruginosastrains with distinct core genome architectures. We showed that these genes were likely acquired from an O12 serotype strain that is closely related toP. aeruginosaPA7. Acquisition and recombination of the “serotype island” resulted in displacement of the native OSA gene cluster and expression of the O12 serotype in the recipients. Serotype switching by recombination has apparently occurred multiple times involving bacteria of various genomic backgrounds. In conclusion, serotype switching in combination with acquisition of an antibiotic resistance determinant most likely contributed to the dissemination of the O12 serotype in clinical settings.IMPORTANCEInfection rates in hospital settings by multidrug-resistant (MDR)Pseudomonas aeruginosaclones have increased during the past decades, and serotype O12 is predominant among these epidemic strains. It is not known why the MDR phenotype is associated with serotype O12 and how this clone type has emerged. This study shows that evolution of MDR O12 strains involved a switch from an ancestral O4 serotype to O12. Serotype switching was the result of horizontal transfer and genetic recombination of lipopolysaccharide (LPS) biosynthesis genes originating from an MDR taxonomic outlierP. aeruginosastrain. Moreover, the recombination event also resulted in acquisition of antibiotic resistance genes. These results impact on our understanding of MDR outbreak strain and serotype evolution and can potentially assist in better monitoring and prevention.

scholarly journals Assessment The Burden of Recent Transmission of M. Tuberculosis Using WGS Analysis In Golmud City In China

2021 ◽  
Author(s):  
Chunfa Liu ◽  
Ping He ◽  
Jiale Fan ◽  
Aijing Ma ◽  
Wencong He ◽  
...  
Keyword(s):  
Transmission Rate ◽  
Previous Treatment ◽  
Epidemiological Data ◽  
Multidrug Resistant ◽  
Population Based ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Resistant Tuberculosis ◽  
Recent Transmission ◽  
Culture Positive

Abstract Background: Whole genome based Mycobacterium tuberculosis (Mtb) surveillance is facilitated to tuberculosis control. The proportion of genotypic clusters in a population represents the recent transmission rate of Mtb.Methods: We did a population based study of culture-positive Mtb in Golmud, Qinghai, China. Whole-genome sequencing (WGS) was used to discriminate the apparent genetic clusters and resistant associated genes of Mtb, and the risk of genomically clustered Mtb was analyzed combined with epidemiological data. Results: A total of 133 cases of culture-positive tuberculosis were recorded between Jan 1, 2013 and Dec 31, 2018, 17 (13%, 17/133) cases of which were multidrug-resistant/rifampicin tuberculosis (MDR/RR-TB). Patients who were previous treatment or were younger than 35 years had high risk of MDR/RR-TB. 62 (47%, 62/132) strains were in 23 genomic clusters that differed by 12 or fewer single nucleotide polymorphisms (SNPs), indicating recent transmission. Patients who were Tibetan nationality, or those 35-44 years old were more likely to have recent transmission. 15 (65%, 15/23) patients with genotypic rifampin resistant tuberculosis have epidemiological link. Mutation of rifampicin resistance associated genes in rpoB Ser450Leu was showed lower cluster rate (42%, 5/12) compared with other mutations.Conclusions: Recent transmissions of Mtb strains, especially genotypic MDR/RR strains, drive the tuberculosis epidemic in Golmud, Qinghai, China.

scholarly journals Development and validation of a Burkholderia pseudomallei core genome multilocus sequence typing scheme to facilitate molecular surveillance

2021 ◽  
Author(s):  
Sabine Lichtenegger ◽  
Trung T. Trinh ◽  
Karoline Assig ◽  
Karola Prior ◽  
Dag Harmsen ◽  
...  
Keyword(s):  
Multilocus Sequence Typing ◽  
Burkholderia Pseudomallei ◽  
Core Genome ◽  
Target Genes ◽  
Severe Disease ◽  
Recent Infection ◽  
Fast Analysis ◽  
Typing Scheme ◽  
Typing Methods ◽  
Good Concordance

Objectives: Burkholderia pseudomallei causes the severe disease melioidosis. Whole genome-sequencing (WGS) based typing methods currently offer the highest resolution for molecular investigations of this genetically diverse pathogen. Still, its routine application in diagnostic laboratories is limited by the need for high computing power, bioinformatic skills and variable bioinformatic approaches, the latter affecting the results. We therefore aimed to establish and validate a WGS-based core genome multilocus sequence typing (cgMLST) scheme, applicable in routine diagnostic settings. Methods: A soft defined core genome was obtained by challenging the B. pseudomallei reference genome K96243 with 469 environmental and clinical genomes, resulting in 4,221 core and 1,359 accessory targets. The scheme was validated with 320 WGS datasets. We compared our novel typing scheme with single nucleotide polymorphism based-approaches investigating closely and distantly related strains. Finally, we applied our scheme for tracking the environmental source of a recent infection. Results: The validation of the scheme detected >95% good cgMLST target genes in 98.4% of the genomes. Comparison with existing typing methods revealed very good concordance. Our scheme proved to be applicable to investigate not only closely related strains, but also the global B. pseudomallei population structure. We successfully utilized our scheme to identify a sugar cane field as the presumable source of a recent melioidosis case. Conclusion: We developed a robust cgMLST typing scheme which integrates high resolution, maximized standardization and fast analysis for the non-bioinformatician. Our typing scheme has the potential to serve as a routinely applicable classification system in B. pseudomallei molecular epidemiology.

scholarly journals Hash-Based Core Genome Multilocus Sequence Typing for Clostridium difficile

2019 ◽  
Vol 58 (1) ◽  
Author(s):  
David W. Eyre ◽  
Tim E. A. Peto ◽  
Derrick W. Crook ◽  
A. Sarah Walker ◽  
Mark H. Wilcox
Keyword(s):  
Clostridium Difficile ◽  
Multilocus Sequence Typing ◽  
Core Genome ◽  
Genetic Relationships ◽  
Nucleotide Polymorphisms ◽  
Content Type ◽  
Recent Transmission ◽  
A Genome ◽  
Performance Penalty

ABSTRACT Pathogen whole-genome sequencing has huge potential as a tool to better understand infection transmission. However, rapidly identifying closely related genomes among a background of thousands of other genomes is challenging. Here, we describe a refinement to core genome multilocus sequence typing (cgMLST) in which alleles at each gene are reproducibly converted to a unique hash, or short string of letters (hash-cgMLST). This avoids the resource-intensive need for a single centralized database of sequentially numbered alleles. We test the reproducibility and discriminatory power of cgMLST/hash-cgMLST compared to those of mapping-based approaches in Clostridium difficile, using repeated sequencing of the same isolates (replicates) and data from consecutive infection isolates from six English hospitals. Hash-cgMLST provided the same results as standard cgMLST, with minimal performance penalty. Comparing 272 replicate sequence pairs using reference-based mapping, there were 0, 1, or 2 single-nucleotide polymorphisms (SNPs) between 262 (96%), 5 (2%), and 1 (<1%) of the pairs, respectively. Using hash-cgMLST, 218 (80%) of replicate pairs assembled with SPAdes had zero gene differences, and 31 (11%), 5 (2%), and 18 (7%) pairs had 1, 2, and >2 differences, respectively. False gene differences were clustered in specific genes and associated with fragmented assemblies, but were reduced using the SKESA assembler. Considering 412 pairs of infections with ≤2 SNPS, i.e., consistent with recent transmission, 376 (91%) had ≤2 gene differences and 16 (4%) had ≥4. Comparing a genome to 100,000 others took <1 min using hash-cgMLST. Hash-cgMLST is an effective surveillance tool for rapidly identifying clusters of related genomes. However, cgMLST/hash-cgMLST generate more false variants than mapping-based approaches. Follow-up mapping-based analyses are likely required to precisely define close genetic relationships.

scholarly journals Development and Application of a Core Genome Multilocus Sequence Typing Scheme for the Health Care-Associated Pathogen Pseudomonas aeruginosa

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Richard A. Stanton ◽  
Gillian McAllister ◽  
Jonathan B. Daniels ◽  
Erin Breaker ◽  
Nicholas Vlachos ◽  
...  
Keyword(s):  
Health Care ◽  
Pseudomonas Aeruginosa ◽  
Multilocus Sequence Typing ◽  
Core Genome ◽  
Health Care Environment ◽  
Content Type ◽  
Health Care Associated Infections ◽  
Opportunistic Human Pathogen ◽  
The Stability ◽  
Core Genes

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen that frequently causes health care-associated infections (HAIs). Due to its metabolic diversity and ability to form biofilms, this Gram-negative nonfermenting bacterium can persist in the health care environment, which can lead to prolonged HAI outbreaks. We describe the creation of a core genome multilocus sequence typing (cgMLST) scheme to provide a stable platform for the rapid comparison of P. aeruginosa isolates using whole-genome sequencing (WGS) data. We used a diverse set of 58 complete P. aeruginosa genomes to curate a set of 4,440 core genes found in each isolate, representing ∼64% of the average genome size. We then expanded the alleles for each gene using 1,991 contig-level genome sequences. The scheme was used to analyze genomes from four historical HAI outbreaks to compare the phylogenies generated using cgMLST to those of other means (traditional MLST, pulsed-field gel electrophoresis [PFGE], and single-nucleotide variant [SNV] analysis). The cgMLST scheme provides sufficient resolution for analyzing individual outbreaks, as well as the stability for comparisons across a variety of isolates encountered in surveillance studies, making it a valuable tool for the rapid analysis of P. aeruginosa genomes.

scholarly journals Comparative genome analysis of a multidrug-resistant Pseudomonas aeruginosa sequence type 277 clone that harbours two copies of the blaSPM-1 gene and multiple single nucleotide polymorphisms in other resistance-associated genes

2019 ◽  
Author(s):  
Ana Paula Barbosa do Nascimento ◽  
Fernando Medeiros Filho ◽  
Hério Sousa ◽  
Hermes Senger ◽  
Rodolpho Mattos Albano ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Resistance ◽  
Single Nucleotide Polymorphisms ◽  
Multidrug Resistant ◽  
Sequence Type ◽  
Genomic Islands ◽  
Genome Comparison ◽  
Nucleotide Polymorphisms ◽  
Emerging Pathogens ◽  
Single Nucleotide

AbstractPseudomonas aeruginosa is one of the most common pathogens related to healthcare-associated infections. The Brazilian isolate, named CCBH4851, is a multidrug-resistant clone belonging to the sequence type 277. The antimicrobial resistance mechanisms of the CCBH4851 strain are associated with the presence of blaSPM-1 gene, encoding a metallo-beta-lactamase, in addition to other exogenously acquired genes. Whole-genome sequencing studies focusing on emerging pathogens are essential to identify physiological key aspects that may lead to the exposure of new targets for therapy. This study was designed to characterize the genome of Pseudomonas aeruginosa CCBH4851 through the detection of genomic features and genome comparison with other Pseudomonas aeruginosa strains. The CCBH4851 closed genome showed features that were consistent with data reported for the specie. However, comparative genomics revealed the absence of genes important for pathogenesis. On the other hand, CCBH4851 genome contained acquired genomic islands that carry additional virulence and antimicrobial resistance-related genes. The presence of single nucleotide polymorphisms in the core genome, mainly those located in resistance-associated genes, suggests that these mutations could influence the multidrug-resistant behavior of CCBH4851. Overall, the characterization of Pseudomonas aeruginosa CCBH4851 complete genome revealed several features that could directly impact the profile of virulence and antibiotic resistance of this pathogen in infectious outbreaks.

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