Epigenetic Response of Yarrowia lipolytica to Stress: Tracking Methylation Level and Search for Methylation Patterns via Whole-Genome Sequencing

DNA methylation is a common, but not universal, epigenetic modification that plays an important role in multiple cellular processes. While definitely settled for numerous plant, mammalian, and bacterial species, the genome methylation in different fungal species, including widely studied and industrially-relevant yeast species, Yarrowia lipolytica, is still a matter of debate. In this paper, we report a differential DNA methylation level in the genome of Y. lipolytica subjected to sequential subculturing and to heat stress conditions. To this end, we adopted repeated batch bioreactor cultivations of Y. lipolytica subjected to thermal stress in specific time intervals. To analyze the variation in DNA methylation between stressed and control cultures, we (a) quantified the global DNA methylation status using an immuno-assay, and (b) studied DNA methylation patterns through whole-genome sequencing. Primarily, we demonstrated that 5 mC modification can be detected using a commercial immuno-assay, and that the modifications are present in Y. lipolytica’s genome at ~0.5% 5 mC frequency. On the other hand, we did not observe any changes in the epigenetic response of Y. lipolytica to heat shock (HS) treatment. Interestingly, we identified a general phenomenon of decreased 5 mC level in Y. lipolytica’s genome in the stationary phase of growth, when compared to a late-exponential epigenome. While this study provides an insight into the subculturing stress response and adaptation to the stress at epigenetic level by Y. lipolytica, it also leaves an open question of inability to detect any genomic DNA methylation level (either in CpG context or context-less) through whole-genome sequencing. The results of ONT sequencing, suggesting that 5 mC modification is either rare or non-existent in Y. lipolytica genome, are contradicted with the results of the immunoassay.

Download Full-text

A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

Scientific Reports ◽

10.1038/s41598-020-80031-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kathy E. Raven ◽

Sophia T. Girgis ◽

Asha Akram ◽

Beth Blane ◽

Danielle Leek ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Dna Extraction ◽

Genome Sequencing ◽

Clinical Microbiology ◽

Bacterial Species ◽

Outbreak Detection ◽

Whole Genome ◽

Library Preparation ◽

Simultaneous Processing ◽

Antimicrobial Resistance Gene

AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

Download Full-text

Transmission of ESBL-producing Enterobacteriaceae and their mobile genetic elements—identification of sources by whole genome sequencing: study protocol for an observational study in Switzerland

BMJ Open ◽

10.1136/bmjopen-2018-021823 ◽

2018 ◽

Vol 8 (2) ◽

pp. e021823 ◽

Cited By ~ 12

Author(s):

Tanja Stadler ◽

Dominik Meinel ◽

Lisandra Aguilar-Bultet ◽

Jana S Huisman ◽

Ruth Schindler ◽

...

Keyword(s):

Observational Study ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Bacterial Species ◽

Mobile Genetic Elements ◽

University Hospital ◽

Whole Genome ◽

Hospital Acquired Infections ◽

Genetic Elements ◽

Hospital Acquired

IntroductionExtended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae were first described in relation with hospital-acquired infections. In the 2000s, the epidemiology of ESBL-producing organisms changed as especially ESBL-producingEscherichia coliwas increasingly described as an important cause of community-acquired infections, supporting the hypothesis that in more recent years ESBL-producing Enterobacteriaceae have probably been imported into hospitals rather than vice versa. Transmission of ESBL-producing Enterobacteriaceae is complicated by ESBL genes being encoded on self-transmissible plasmids, which can be exchanged among the same and different bacterial species. The aim of this research project is to quantify hospital-wide transmission of ESBL-producing Enterobacteriaceae on both the level of bacterial species and the mobile genetic elements and to determine if hospital-acquired infections caused by ESBL producers are related to strains and mobile genetic elements predominantly circulating in the community or in the healthcare setting. This distinction is critical in prevention since the former emphasises the urgent need to establish or reinforce antibiotic stewardship programmes, and the latter would call for more rigorous infection control.Methods and analysisThis protocol presents an observational study that will be performed at the University Hospital Basel and in the city of Basel, Switzerland. ESBL-producing Enterobacteriaceae will be collected from any specimens obtained by routine clinical practice or by active screening in both inpatient and outpatient settings, as well as from wastewater samples and foodstuffs, both collected monthly over a 12-month period for analyses by whole genome sequencing. Bacterial chromosomal, plasmid and ESBL-gene sequences will be compared within the cohort to determine genetic relatedness and migration between humans and their environment.Ethics and disseminationThis study has been approved by the local ethics committee (Ethikkommission Nordwest-und Zentralschweiz) as a quality control project (Project-ID 2017–00100). The results of this study will be published in peer-reviewed medical journals, communicated to participants, the general public and all relevant stakeholders.

Download Full-text

Spill-Over from Public Health? First Detection of an OXA-48-Producing Escherichia coli in a German Pig Farm

Microorganisms ◽

10.3390/microorganisms8060855 ◽

2020 ◽

Vol 8 (6) ◽

pp. 855 ◽

Cited By ~ 2

Author(s):

Alexandra Irrgang ◽

Natalie Pauly ◽

Bernd-Alois Tenhagen ◽

Mirjam Grobbel ◽

Annemarie Kaesbohrer ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Bacterial Species ◽

Multidrug Resistant ◽

Meat Production ◽

Whole Genome ◽

Phenotypic Resistance ◽

Long Read ◽

Pig Farm ◽

Human Infections

Resistance to carbapenems is a severe threat to human health. These last resort antimicrobials are indispensable for the treatment of severe human infections with multidrug-resistant Gram-negative bacteria. In accordance with their increasing medical impact, carbapenemase-producing Enterobacteriaceae (CPE) might be disseminated from colonized humans to non-human reservoirs (i.e., environment, animals, food). In Germany, the occurrence of CPE in livestock and food has been systematically monitored since 2016. In the 2019 monitoring, an OXA-48-producing E. coli (19-AB01443) was recovered from a fecal sample of a fattening pig. Phenotypic resistance was confirmed by broth microdilution and further characterized by PFGE, conjugation, and combined short-/long-read whole genome sequencing. This is the first detection of this resistance determinant in samples from German meat production. Molecular characterization and whole-genome sequencing revealed that the blaOXA-48 gene was located on a common pOXA-48 plasmid-prototype. This plasmid-type seems to be globally distributed among various bacterial species, but it was frequently associated with clinical Klebsiella spp. isolates. Currently, the route of introduction of this plasmid/isolate combination into the German pig production is unknown. We speculate that due to its strong correlation with human isolates a transmission from humans to livestock has occurred.

Download Full-text

DNA methylation patterns of β-globin cluster in β-thalassemia patients

Clinical Epigenetics ◽

10.1186/s13148-020-00987-2 ◽

2020 ◽

Vol 12 (1) ◽

Author(s):

Xiuqin Bao ◽

Yangjin Zuo ◽

Diyu Chen ◽

Cunyou Zhao

Keyword(s):

Dna Methylation ◽

Cord Blood ◽

Methylation Level ◽

Epigenetic Modification ◽

Fetal Hemoglobin ◽

Cpg Sites ◽

Hbf Level ◽

Globin Promoter ◽

Methylation Patterns ◽

Normal Controls

Abstract Background Reactivation of fetal hemoglobin (HbF, α2γ2) holds a therapeutic target for β-thalassemia and sickle cell disease. Although many HbF regulators have been identified, the methylation patterns in β-globin cluster driving the fetal-to-adult hemoglobin switch remains to be determined. Results Here, we evaluated DNA methylation patterns of the β-globin cluster from peripheral bloods of 105 β0/β0 thalassemia patients and 44 normal controls. We also recruited 15 bone marrows and 4 cord blood samples for further evaluation. We identified that the CpG sites in the locus control region (LCR) DNase I hypersensitive site 4 and 3 (HS4-3) regions, and γ- and β-globin promoters displayed hypomethylation in β0/β0-thalassemia patients, especially for the patients with high HbF level, as compared with normal controls. Furthermore, hypomethylations in most of CpG sites of the HS4-3 core regions were also observed in bone marrows (BM) of β0/β0-patients compared with normal controls; and methylation level of γ-globin promoter -50 and + 17 CpG sites showed lower methylation level in patients with high HbF level compared with those with low HbF level and a negative correlation with HbF level among β0-thalassemia patients. Finally, γ-globin promoter + 17 and + 50 CpG sites also displayed significant hypomethylation in cord blood (CB) tissues compared with BM tissues from normal controls. Conclusions Our findings revealed methylation patterns in β-globin cluster associated with β0 thalassemia disease and γ-globin expression, contributed to understand the epigenetic modification in β0 thalassemia patients and provided candidate targets for the therapies of β-hemoglobinopathies.

Download Full-text

Food Workers as a Reservoir of Extended-Spectrum-Cephalosporin-Resistant Salmonella Strains in Japan

Applied and Environmental Microbiology ◽

10.1128/aem.00072-20 ◽

2020 ◽

Vol 86 (13) ◽

Author(s):

Hiroaki Shigemura ◽

Eri Sakatsume ◽

Tsuyoshi Sekizuka ◽

Hiroshi Yokoyama ◽

Kunihiko Hamada ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Bacterial Species ◽

Diffusion Method ◽

Whole Genome ◽

Disk Diffusion Method ◽

Content Type ◽

Extended Spectrum ◽

Food Workers ◽

M 14

ABSTRACT Dissemination of extended-spectrum-cephalosporin (ESC)-resistant Salmonella, especially extended-spectrum-β-lactamase (ESBL)-producing Salmonella, is a concern worldwide. Here, we assessed Salmonella carriage by food workers in Japan to clarify the prevalence of ESC-resistant Salmonella harboring blaCTX-M. We then characterized the genetic features, such as transposable elements, of blaCTX-M-harboring plasmids using whole-genome sequencing. A total of 145,220 stool samples were collected from food workers, including cooks and servers from several restaurants, as well as food factory workers, from January to October 2017. Isolated salmonellae were subjected to antimicrobial susceptibility testing (disk diffusion method), and whole-genome sequencing was performed for Salmonella strains harboring blaCTX-M. Overall, 164 Salmonella isolates (0.113%) were recovered from 164 samples, from which we estimated that at least 0.113% (95% confidence interval [CI]: 0.096 to 0.132%) of food workers may carry Salmonella. Based on this estimation, 3,473 (95% CI = 2,962 to 4,047) individuals among the 3,075,330 Japanese food workers are likely to carry Salmonella. Of the 158 culturable isolates, seven showed resistance to ESCs: three isolates harbored blaCMY-2 and produced AmpC β-lactamase, while four ESBL-producing isolates harbored blaCTX-M-14 (n = 1, Salmonella enterica serovar Senftenberg) or blaCTX-M-15 (n = 3, S. enterica serovar Haardt). blaCTX-M-15 was chromosomally located in the S. Haardt isolates, which also contained ISEcp1, while the S. Senftenberg isolate contained an IncFIA(HI1)/IncHI1A/IncHI1B(R27) hybrid plasmid carrying blaCTX-M-14 along with ISEcp1. This study indicates that food workers may be a reservoir of ESBL-producing Salmonella and associated genes. Thus, these workers may contribute to the spread of blaCTX-M via plasmids or mobile genetic elements such as ISEcp1. IMPORTANCE Antimicrobial-resistant Salmonella bacteria arise in farm environments through imprudent use of antimicrobials. Subsequently, these antimicrobial-resistant strains, such as extended-spectrum-β-lactamase (ESBL)-producing Salmonella, may be transmitted to humans via food animal-derived products. Here, we examined Salmonella carriage among food handlers in Japan. Overall, 164 of 145,220 fecal samples (0.113%) were positive for Salmonella. Among the 158 tested isolates, four were identified as ESBL-producing isolates carrying ESBL determinants blaCTX-M-15 or blaCTX-M-14. In all cases, the genes coexisted with ISEcp1, regardless of whether they were located on the chromosome or on a plasmid. Our findings suggest that food workers may be a reservoir of ESBL-producing strains and could contribute to the spread of resistance genes from farm-derived Salmonella to other bacterial species present in the human gut.

Download Full-text

Nonsynonymous Polymorphism Counts in Bacterial Genomes: a Comparative Examination

Applied and Environmental Microbiology ◽

10.1128/aem.02002-20 ◽

2020 ◽

Vol 87 (1) ◽

Author(s):

Sara L. Loo ◽

Anna Ong ◽

Wunna Kyaw ◽

Loïc M. Thibaut ◽

Ruiting Lan ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Negative Selection ◽

Bacterial Species ◽

Host Population ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Chronic Infections ◽

Snp Data ◽

Sequencing Studies

ABSTRACT Genomic data reveal single-nucleotide polymorphisms (SNPs) that may carry information about the evolutionary history of bacteria. However, it remains unclear what inferences about selection can be made from genomic SNP data. Bacterial species are often sampled during epidemic outbreaks or within hosts during the course of chronic infections. SNPs obtained from genomic analysis of these data are not necessarily fixed. Treating them as fixed during analysis by using measures such as the ratio of nonsynonymous to synonymous evolutionary changes (dN/dS) may lead to incorrect inferences about the strength and direction of selection. In this study, we consider data from a range of whole-genome sequencing studies of bacterial pathogens and explore patterns of nonsynonymous variation to assess whether evidence of selection can be identified by investigating SNP counts alone across multiple WGS studies. We visualize these SNP data in ways that highlight their relationship to neutral baseline expectations. These neutral expectations are based on a simple model of mutation, from which we simulate SNP accumulation to investigate how SNP counts are distributed under alternative assumptions about positive and negative selection. We compare these patterns with empirical SNP data and illustrate the general difficulty of detecting positive selection from SNP data. Finally, we consider whether SNP counts observed at the between-host population level diﬀer from those observed at the within-host level and find some evidence that suggests that dynamics across these two scales are driven by diﬀerent underlying processes. IMPORTANCE Identifying selection from SNP data obtained from whole-genome sequencing studies is challenging. Some current measures used to identify and quantify selection acting on genomes rely on fixed diﬀerences; thus, these are inappropriate for SNP data where variants are not fixed. With the increase in whole-genome sequencing studies, it is important to consider SNP data in the context of evolutionary processes. How SNPs are counted and analyzed can help in understanding mutation accumulation and trajectories of strains. We developed a tool for identifying possible evidence of selection and for comparative analysis with other SNP data. We propose a model that provides a rule-of-thumb guideline and two new visualization techniques that can be used to interpret and compare SNP data. We quantify the expected proportion of nonsynonymous SNPs in coding regions under neutrality and demonstrate its use in identifying evidence of positive and negative selection from simulations and empirical data.

Download Full-text

Complete Genome Sequences of 12 Species of Stable Defined Moderately Diverse Mouse Microbiota 2: TABLE 1

Genome Announcements ◽

10.1128/genomea.00951-16 ◽

2016 ◽

Vol 4 (5) ◽

Cited By ~ 24

Author(s):

Yasuhiro Uchimura ◽

Madeleine Wyss ◽

Sandrine Brugiroux ◽

Julien P. Limenitakis ◽

Bärbel Stecher ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Complete Genome ◽

Bacterial Species ◽

Whole Genome ◽

Genome Sequences ◽

Pacbio Sequencing ◽

Sequencing Platform ◽

Germ Free

We report here the complete genome sequences of 12 bacterial species of stable defined moderately diverse mouse microbiota 2 (sDMDMm2) used to colonize germ-free mice with defined microbes. Whole-genome sequencing of these species was performed using the PacBio sequencing platform yielding circularized genome sequences of all 12 species.

Download Full-text

Whole-Genome Sequencing for Molecular Characterization of Carbapenem-Resistant Enterobacteriaceae Causing Lower Urinary Tract Infection among Pediatric Patients

Antibiotics ◽

10.3390/antibiotics10080972 ◽

2021 ◽

Vol 10 (8) ◽

pp. 972

Author(s):

Hassan Al Mana ◽

Sathyavathi Sundararaju ◽

Clement K. M. Tsui ◽

Andres Perez-Lopez ◽

Hadi Yassine ◽

...

Keyword(s):

Urinary Tract ◽

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Bacterial Species ◽

Whole Genome ◽

E Coli ◽

Carbapenem Resistant ◽

Tract Infections ◽

Carbapenem Resistant Enterobacteriaceae

Antibiotic resistance is a growing public health problem globally, incurring health and cost burdens. The occurrence of antibiotic-resistant bacterial infections has increased significantly over the years. Gram-negative bacteria display the broadest resistance range, with bacterial species expressing extended-spectrum β-lactamases (ESBLs), AmpC, and carbapenemases. All carbapenem-resistant Enterobacteriaceae (CRE) isolates from pediatric urinary tract infections (UTIs) between October 2015 and November 2019 (n = 30). All isolates underwent antimicrobial resistance phenotypic testing using the Phoenix NMIC/ID-5 panel, and carbapenemase production was confirmed using the NG-Test CARBA 5 assay. Whole-genome sequencing was performed on the CREs. The sequence type was identified using the Achtman multi-locus sequence typing scheme, and antimicrobial resistance markers were identified using ResFinder and the CARD database. The most common pathogens causing CRE UTIs were E. coli (63.3%) and K. pneumoniae (30%). The most common carbapenemases produced were OXA-48-like enzymes (46.6%) and NDM enzymes (40%). Additionally, one E. coli harbored IMP-26, and two K. pneumoniae possessed mutations in ompK37 and/or ompK36. Lastly, one E. coli had a mutation in the marA porin and efflux pump regulator. The findings highlight the difference in CRE epidemiology in the pediatric population compared to Qatar’s adult population, where NDM carbapenemases are more common.

Download Full-text

Haemophilus to meningococci transfer of beta-lactamase

10.1101/308056 ◽

2018 ◽

Author(s):

Eva Hong ◽

Ala-Eddine Deghmane ◽

Muhamed-Kheir Taha

Keyword(s):

Haemophilus Influenzae ◽

Whole Genome Sequencing ◽

Effective Treatment ◽

Genome Sequencing ◽

Meningococcal Disease ◽

Horizontal Transfer ◽

Bacterial Species ◽

Whole Genome ◽

Beta Lactamase ◽

Beta Lactamases

AbstractWe report the detection in France of a beta-lactamase producing invasive meningococcal isolate. Whole genome sequencing of the isolate revealed ROB-1 type beta-lactamase that is frequently encountered in Haemophilus influenzae suggesting horizontal transfer between isolates of these bacterial species. Beta-lactamases are exceptional in meningococci with no reports from more than two decades. This report is worrying as the expansion of such isolates may jeopardize the effective treatment against invasive meningococcal disease.

Download Full-text

Whole-Genome Sequencing and Annotation of Clostridium tyrobutyricum Strain Cirm BIA 2237, Isolated from Silage

Microbiology Resource Announcements ◽

10.1128/mra.00492-19 ◽

2019 ◽

Vol 8 (30) ◽

Author(s):

Edouard Munier ◽

Hélène Licandro-Séraut ◽

Christine Achilleos ◽

Rémy Cachon ◽

Eric Beuvier

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Complete Genome ◽

Bacterial Species ◽

Whole Genome ◽

Clostridium Tyrobutyricum ◽

Content Type ◽

Complete Genome Sequencing

Clostridium tyrobutyricum is the main bacterial species leading to the late blowing defect, a major cause of spoilage in semihard and hard cheeses. This study reports the complete genome sequencing, assembly, and annotation of C. tyrobutyricum strain Cirm BIA 2237, formerly called CNRZ 608, isolated from silage.

Download Full-text