Whole-Genome Sequencing for Predicting Clarithromycin Resistance in Mycobacterium abscessus

ABSTRACT Mycobacterium abscessus is emerging as an important pathogen in chronic lung diseases, with concern regarding patient-to-patient transmission. The recent introduction of routine whole-genome sequencing (WGS) as a replacement for existing reference techniques in England provides an opportunity to characterize the genetic determinants of resistance. We conducted a systematic review to catalogue all known resistance-determining mutations. This knowledge was used to construct a predictive algorithm based on mutations in the erm(41) and rrl genes which was tested on a collection of 203 sequentially acquired clinical isolates for which there were paired genotype/phenotype data. A search for novel resistance-determining mutations was conducted using a heuristic algorithm. The sensitivity of existing knowledge for predicting resistance in clarithromycin was 95% (95% confidence interval [CI], 89 to 98%), and the specificity was 66% (95% CI, 54 to 76%). The subspecies alone was a poor predictor of resistance to clarithromycin. Eight potential new resistance-conferring single nucleotide polymorphisms (SNPs) were identified. WGS demonstrated probable resistance-determining SNPs in regions that the NTM-DR line probe cannot detect. These mutations are potentially clinically important, as they all occurred in samples that were predicted to be inducibly resistant and for which a macrolide would therefore currently be indicated. We were unable to explain all resistance, raising the possibility of the involvement of other as yet unidentified genes.

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Whole genome sequencing for predicting clarithromycin resistance in Mycobacterium abscessus

10.1101/251918 ◽

2018 ◽

Cited By ~ 1

Author(s):

Samuel Lipworth ◽

Natasha Hough ◽

Laura Leach ◽

Marcus Morgan ◽

Katie Jeffery ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Lung Diseases ◽

Mycobacterium Abscessus ◽

Whole Genome ◽

Genetic Determinants ◽

Predictive Algorithm ◽

Chronic Lung Diseases ◽

Clarithromycin Resistance ◽

Phenotype Data

AbstractMycobacterium abscessus is emerging as an important pathogen in chronic lung diseases with concern regarding patient to patient transmission. The recent introduction of routine whole genome sequencing (WGS) as a replacement for existing reference techniques in England provides an opportunity to characterise the genetic determinants of resistance. We conducted a systematic review to catalogue all known resistance determining mutations. This knowledge was used to construct a predictive algorithm based on mutations in the erm(41) and rrl genes which was tested on a collection of 203 sequentially acquired clinical isolates for which there was paired genotype/phenotype data. A search for novel resistance determining mutations was conducted using an heuristic algorithm.The sensitivity of existing knowledge for predicting resistance in clarithromycin was 95% (95% CI 89 – 98%) and the specificity was 66% (95% CI 54 – 76%). Subspecies alone was a poor predictor of resistance to clarithromycin. Eight potential new resistance conferring SNPs were identified. WGS demonstrates probable resistance determining SNPs in regions the NTM-DR line probe cannot detect. These mutations are potentially clinically important as they all occurred in samples predicted to be inducibly resistant, and for which a macrolide would therefore currently be indicated. We were unable to explain all resistance, raising the possibility of the involvement of other as yet unidentified genes.

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Whole-Genome Sequencing Allows for Improved Identification of Persistent Listeria monocytogenes in Food-Associated Environments

Applied and Environmental Microbiology ◽

10.1128/aem.01049-15 ◽

2015 ◽

Vol 81 (17) ◽

pp. 6024-6037 ◽

Cited By ~ 76

Author(s):

Matthew J. Stasiewicz ◽

Haley F. Oliver ◽

Martin Wiedmann ◽

Henk C. den Bakker

Keyword(s):

Listeria Monocytogenes ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome Sequence ◽

Whole Genome ◽

Genetic Determinants ◽

Single Nucleotide ◽

Content Type ◽

Food Borne ◽

Clonal Spread

ABSTRACTWhile the food-borne pathogenListeria monocytogenescan persist in food associated environments, there are no whole-genome sequence (WGS) based methods to differentiate persistent from sporadic strains. Whole-genome sequencing of 188 isolates from a longitudinal study ofL. monocytogenesin retail delis was used to (i) apply single-nucleotide polymorphism (SNP)-based phylogenetics for subtyping ofL. monocytogenes, (ii) use SNP counts to differentiate persistent from repeatedly reintroduced strains, and (iii) identify genetic determinants ofL. monocytogenespersistence. WGS analysis revealed three prophage regions that explained differences between three pairs of phylogenetically similar populations with pulsed-field gel electrophoresis types that differed by ≤3 bands. WGS-SNP-based phylogenetics found that putatively persistentL. monocytogenesrepresent SNP patterns (i) unique to a single retail deli, supporting persistence within the deli (11 clades), (ii) unique to a single state, supporting clonal spread within a state (7 clades), or (iii) spanning multiple states (5 clades). Isolates that formed one of 11 deli-specific clades differed by a median of 10 SNPs or fewer. Isolates from 12 putative persistence events had significantly fewer SNPs (median, 2 to 22 SNPs) than between isolates of the same subtype from other delis (median up to 77 SNPs), supporting persistence of the strain. In 13 events, nearly indistinguishable isolates (0 to 1 SNP) were found across multiple delis. No individual genes were enriched among persistent isolates compared to sporadic isolates. Our data show that WGS analysis improves food-borne pathogen subtyping and identification of persistent bacterial pathogens in food associated environments.

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Whole Genome Sequencing of Nontuberculous Mycobacterium (NTM) Isolates from Sputum and Environmental Specimens in Co-Habiting Patients with NTM Pulmonary Disease

10.21203/rs.2.17722/v1 ◽

2019 ◽

Author(s):

Jung-Ki Yoon ◽

Taek Soo Kim ◽

Jong-Il Kim ◽

Jae-Joon Yim

Keyword(s):

Whole Genome Sequencing ◽

Pulmonary Disease ◽

Genome Sequencing ◽

Genetic Distances ◽

Mycobacterium Abscessus ◽

Nontuberculous Mycobacterium ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Next Generation Sequencing Ngs

Abstract Background : Nontuberculous mycobacterium (NTM) species are ubiquitous microorganisms. NTM pulmonary disease (NTM-PD) is caused not by human-to-human transmission but by independent environmental acquisition. However, recent studies using next-generation sequencing (NGS) have reported trans-continental spread of Mycobacterium abscessus among patients with cystic fibrosis. Results : We investigated NTM genomes through NGS to examine transmission patterns in three pairs of co-habiting NTM-PD patients who were suspected of patient-to-patient transmission. Three pairs of patients with NTM-PD co-habiting for at least 15 years were enrolled: a mother and a daughter with M. avium PD, a couple with M. intracellulare PD, and a second couple, one of whom was infected with M. intracellulare PD and the other of whom was infected with M. abscessus subsp. massiliense PD. Whole genome sequencing was performed using NTM colonies isolated from patients and environmental specimens. Genetic distances were estimated based on single nucleotide polymorphisms (SNPs) in the NTM genomes. Comparing SNPs in the consensus regions, the minimum pairwise SNP distances of NTM isolates derived from the two pairs of patients infected with the same NTM species were over 10,000. In phylogenetic analysis, the NTM isolates from patients with M. avium PD clustered with isolates from different environmental sources. Conclusions : In conclusion, considering the genetic distances between NTM strains, the likelihood of patient-to-patient transmission in pairs of co-habiting NTM-PD patients without overt immune deficiency is minimal.

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Whole Genome Sequencing of Nontuberculous Mycobacterium (NTM) Isolates from Sputum Specimens of Co-Habiting Patients with NTM Pulmonary Disease and NTM Isolates from Their Environment

10.21203/rs.2.17722/v3 ◽

2020 ◽

Author(s):

Jung-Ki Yoon ◽

Taek Soo Kim ◽

Jong-Il Kim ◽

Jae-Joon Yim

Keyword(s):

Whole Genome Sequencing ◽

Pulmonary Disease ◽

Genome Sequencing ◽

Genetic Distances ◽

Mycobacterium Abscessus ◽

Nontuberculous Mycobacterium ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Next Generation Sequencing Ngs

Abstract Background: Nontuberculous mycobacterium (NTM) species are ubiquitous microorganisms. NTM pulmonary disease (NTM-PD) is thought to be caused not by human-to-human transmission but by independent environmental acquisition. However, recent studies using next-generation sequencing (NGS) have reported trans-continental spread of Mycobacterium abscessus among patients with cystic fibrosis. Results: We investigated NTM genomes through NGS to examine transmission patterns in three pairs of co-habiting patients with NTM-PD who were suspected of patient-to-patient transmission. Three pairs of patients with NTM-PD co-habiting for at least 15 years were enrolled: a mother and a daughter with M. avium-PD, a couple with M. intracellulare-PD, and a second couple, one of whom was infected with M. intracellulare and the other of whom was infected with M. abscessus. Whole genome sequencing was performed using patients’ NTM isolates as well as environmental specimens. Genetic distances were estimated based on single nucleotide polymorphisms (SNPs). By comparison with the genetic distances among 78 publicly available NTM genomes, NTM isolates derived from the two pairs of patients infected with the same NTM species were not closely related to each other. In phylogenetic analysis, the NTM isolates from patients with M. avium-PD clustered with isolates from different environmental sources.Conclusions: In conclusion, considering the genetic distances between NTM strains, the likelihood of patient-to-patient transmission in pairs of co-habiting NTM-PD patients without overt immune deficiency is minimal.

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Utility of Whole-Genome Sequencing in Characterizing Acinetobacter Epidemiology and Analyzing Hospital Outbreaks

Journal of Clinical Microbiology ◽

10.1128/jcm.01818-15 ◽

2015 ◽

Vol 54 (3) ◽

pp. 593-612 ◽

Cited By ~ 44

Author(s):

Margaret A. Fitzpatrick ◽

Egon A. Ozer ◽

Alan R. Hauser

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Phylogenetic Trees ◽

Acinetobacter Calcoaceticus ◽

Whole Genome ◽

Strain Typing ◽

Nucleotide Polymorphisms ◽

Content Type ◽

Carbapenem Resistant ◽

Outbreak Investigations

Acinetobacter baumanniifrequently causes nosocomial infections and outbreaks. Whole-genome sequencing (WGS) is a promising technique for strain typing and outbreak investigations. We compared the performance of conventional methods with WGS for strain typing clinicalAcinetobacterisolates and analyzing a carbapenem-resistantA. baumannii(CRAB) outbreak. We performed two band-based typing techniques (pulsed-field gel electrophoresis and repetitive extragenic palindromic-PCR), multilocus sequence type (MLST) analysis, and WGS on 148Acinetobacter calcoaceticus-A. baumanniicomplex bloodstream isolates collected from a single hospital from 2005 to 2012. Phylogenetic trees inferred from core-genome single nucleotide polymorphisms (SNPs) confirmed threeAcinetobacterspecies within this collection. Four majorA. baumanniiclonal lineages (as defined by MLST) circulated during the study, three of which are globally distributed and one of which is novel. WGS indicated that a threshold of 2,500 core SNPs accurately distinguishedA. baumanniiisolates from different clonal lineages. The band-based techniques performed poorly in assigning isolates to clonal lineages and exhibited little agreement with sequence-based techniques. After applying WGS to a CRAB outbreak that occurred during the study, we identified a threshold of 2.5 core SNPs that distinguished nonoutbreak from outbreak strains. WGS was more discriminatory than the band-based techniques and was used to construct a more accurate transmission map that resolved many of the plausible transmission routes suggested by epidemiologic links. Our study demonstrates that WGS is superior to conventional techniques forA. baumanniistrain typing and outbreak analysis. These findings support the incorporation of WGS into health care infection prevention efforts.

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Loss and Gain in the Evolution of theSalmonella entericaSerovar Gallinarum Biovar Pullorum Genome

mSphere ◽

10.1128/msphere.00627-18 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 6

Author(s):

Yachen Hu ◽

Zhenyu Wang ◽

Bin Qiang ◽

Yaohui Xu ◽

Xiang Chen ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genomic Analysis ◽

Developed Countries ◽

Epidemiological Surveillance ◽

Recent Common Ancestor ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Content Type ◽

Most Recent Common Ancestor

ABSTRACTSalmonella entericasubspeciesentericaserovar Gallinarum biovar Pullorum (S. Pullorum) is the etiological agent of pullorum disease, causing white diarrhea with high mortality in chickens. There are many unsolved issues surrounding the epidemiology ofS. Pullorum, including its origin and transmission history as well as the discordance between its phenotypic heterogeneity and genetic monomorphism. In this paper, we report the results of whole-genome sequencing of a panel of 97S. Pullorum strains isolated between 1962 and 2014 from four countries across three continents. We utilized 6,795 core genome single nucleotide polymorphisms (SNPs) to reconstruct a phylogenetic tree within a spatiotemporal Bayesian framework, estimating that the most recent common ancestor ofS. Pullorum emerged in ∼914 CE (95% confidence interval [95%CI], 565 to 1273 CE). The extantS. Pullorum strains can be divided into four distinct lineages, each of which is significantly associated with geographical distribution. The intercontinental transmissions of lineages III and IV can be traced to the mid-19th century and are probably related to the “Hen Fever” prevalent at that time. Further genomic analysis indicated that the loss or pseudogenization of functional genes involved in metabolism and virulence inS. Pullorum has been ongoing since before and after divergence from the ancestor. In contrast, multiple prophages and plasmids have been acquired byS. Pullorum, and these have endowed it with new characteristics, especially the multidrug resistance conferred by two large plasmids in lineage I. The results of this study provide insight into the evolution ofS. Pullorum and prove the efficiency of whole-genome sequencing in epidemiological surveillance of pullorum disease.IMPORTANCEPullorum disease, an acute poultry septicemia caused bySalmonellaGallinarum biovar Pullorum, is fatal for young chickens and is a heavy burden on poultry industry. The pathogen is rare in most developed countries but still extremely difficult to eliminate in China. Efficient epidemiological surveillance necessitates clarifying the origin of the isolates from different regions and their phylogenic relationships. Genomic epidemiological analysis of 97S. Pullorum strains was carried out to reconstruct the phylogeny and transmission history ofS. Pullorum. Further analysis demonstrated that functional gene loss and acquisition occurred simultaneously throughout the evolution ofS. Pullorum, both of which reflected adaptation to the changing environment. The result of our study will be helpful in surveillance and prevention of pullorum disease.

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Salmonella entericaPhylogeny Based on Whole-Genome Sequencing Reveals Two New Clades and Novel Patterns of Horizontally Acquired Genetic Elements

mBio ◽

10.1128/mbio.02303-18 ◽

2018 ◽

Vol 9 (6) ◽

Cited By ~ 24

Author(s):

Jay Worley ◽

Jianghong Meng ◽

Marc W. Allard ◽

Eric W. Brown ◽

Ruth E. Timme

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Foodborne Pathogens ◽

Bacterial Species ◽

Whole Genome Sequence ◽

Nucleotide Sequencing ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Content Type ◽

Genetic Elements

ABSTRACTUsing whole-genome sequence (WGS) data from the GenomeTrakr network, a globally distributed network of laboratories sequencing foodborne pathogens, we present a new phylogeny ofSalmonella entericacomprising 445 isolates from 266 distinct serovars and originating from 52 countries. This phylogeny includes two previously unidentifiedS. entericasubsp.entericaclades. Serovar Typhi is shown to be nested within clade A. Our findings are supported by both phylogenetic support, based on a core genome alignment, and Bayesian approaches, based on single-nucleotide polymorphisms. Serovar assignments were refined byin silicoanalysis using SeqSero. More than 10% of serovars were either polyphyletic or paraphyletic. We found variable genetic content in these isolates relating to gene mobilization and virulence factors which have different distributions within clades. Gifsy-1- and Gifsy-2-like phages appear more prevalent in clade A; other viruses are more evenly distributed. Our analyses reveal IncFII is the predominant plasmid replicon inS. enterica. Few core or clade-defining virulence genes are observed, and their distributions appear probabilistic in nature. Together, these patterns demonstrate that genetic exchange withinS. entericais more extensive and frequent than previously realized, which significantly alters how we view the genetic structure of the bacterial species.IMPORTANCERapid improvements in nucleotide sequencing access and affordability have led to a drastic increase in availability of genetic information. This information will improve the accuracy of molecular descriptions, including serovars, withinS. enterica. Although the concept of serovars continues to be useful, it may have more significant limitations than previously understood. Furthermore, the discrete absence or presence of specific genes can be an unstable indicator of phylogenetic identity. Whole-genome sequencing provides more rigorous tools for assessing the distributions of these genes. Our phylogenetic and genetic content analyses reveal how active genetic elements are dynamically distributed within a species, allowing us to better understand genetic reservoirs and underlying bacterial evolution.

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Whole Genome Sequencing of Nontuberculous Mycobacterium (NTM) Isolates from Sputum Specimens of Co-Habiting Patients with NTM Pulmonary Disease and NTM Isolates from Their Environment

10.21203/rs.2.17722/v2 ◽

2020 ◽

Author(s):

Jung-Ki Yoon ◽

Taek Soo Kim ◽

Jong-Il Kim ◽

Jae-Joon Yim

Keyword(s):

Whole Genome Sequencing ◽

Pulmonary Disease ◽

Genome Sequencing ◽

Genetic Distances ◽

Mycobacterium Abscessus ◽

Nontuberculous Mycobacterium ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Next Generation Sequencing Ngs

Abstract Background : Nontuberculous mycobacterium (NTM) species are ubiquitous microorganisms. NTM pulmonary disease (NTM-PD) is caused not by human-to-human transmission but by independent environmental acquisition. However, recent studies using next-generation sequencing (NGS) have reported trans-continental spread of Mycobacterium abscessus among patients with cystic fibrosis. Results : We investigated NTM genomes through NGS to examine transmission patterns in three pairs of co-habiting patients with NTM-PD who were suspected of patient-to-patient transmission. Three pairs of patients with NTM-PD co-habiting for at least 15 years were enrolled: a mother and a daughter with M. avium- PD, a couple with M. intracellulare- PD, and a second couple, one of whom was infection with M. intracellulare and the other of whom was infected with M. abscessus . Whole genome sequencing was performed using patients’ NTM isolates as well as environmental specimens. Genetic distances were estimated based on single nucleotide polymorphisms (SNPs). By comparison with the genetic distances among 78 publicly available NTM genomes, NTM isolates derived from the two pairs of patients infected with the same NTM species were not closely related to each other. In phylogenetic analysis, the NTM isolates from patients with M. avium -PD clustered with isolates from different environmental sources. Conclusions : In conclusion, considering the genetic distances between NTM strains, the likelihood of patient-to-patient transmission in pairs of co-habiting NTM-PD patients without overt immune deficiency is minimal.

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Whole-Genome Sequencing and Bioinformatic Analysis of Isolates from Foodborne Illness Outbreaks ofCampylobacter jejuniandSalmonella enterica

Journal of Clinical Microbiology ◽

10.1128/jcm.00161-18 ◽

2018 ◽

Vol 56 (11) ◽

Cited By ~ 9

Author(s):

Kelly F. Oakeson ◽

Jennifer Marie Wagner ◽

Andreas Rohrwasser ◽

Robyn Atkinson-Dunn

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Raw Milk ◽

Bioinformatic Analysis ◽

Foodborne Illness ◽

Reference Sequence ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Content Type ◽

Evolutionary Relatedness

ABSTRACTWhole-genome sequencing (WGS) via next-generation sequencing (NGS) technologies is a powerful tool for determining the relatedness of bacterial isolates in foodborne illness detection and outbreak investigations. WGS has been applied to national outbreaks (for example,Listeria monocytogenes); however, WGS has rarely been used in smaller local outbreaks. The current study demonstrates the superior resolution of genetic and evolutionary relatedness generated by WGS data analysis, compared to pulsed-field gel electrophoresis (PFGE). The current study retrospectively applies WGS and a reference-free bioinformatic analysis to a Utah-specific outbreak ofCampylobacter jejuniassociated with raw milk and to a national multistate outbreak ofSalmonella entericasubsp.entericaserovar Typhimurium associated with rotisserie chicken, both of which were characterized previously by PFGE. Together, these analyses demonstrate how a reference-free WGS workflow is not reliant on determination of a reference sequence, like WGS workflows that are based on single-nucleotide polymorphisms, or the need for curated allele databases, like multilocus sequence typing workflows.

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