Deciphering the pathogenic role of rare RAF1 heterozygous missense mutation in the late-presenting DDH.

Background: Developmental Dysplasia of the Hip (DDH) is a skeletal disorder where late-presenting forms often escape early diagnosis, leading to limb and pain in adults. The genetic basis of DDH is not fully understood despite known genetic predispositions. Methods: We employed Whole Genome Sequencing (WGS) to explore the genetic factors in late-presenting DDH in two unrelated families, supported by phenotypic analyses and in vitro validation. Results: In both cases, a novel de novo heterozygous missense mutation in RAF1 (c.193A>G [p.Lys65Glu]) was identified. This mutation impacted RAF1 protein structure and function, altering downstream signaling in the Ras/ERK pathway, as demonstrated by bioinformatics, molecular dynamics simulations, and in vitro validations. Conclusion: This study contributes to our understanding of the genetic factors involved in DDH by identifying a novel mutation in RAF1. The identification of the RAF1 mutation suggests a possible involvement of the Ras/ERK pathway in the pathogenesis of late-presenting DDH, indicating its potential role in skeletal development.

Genomic characterization of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) strains circulating in three university hospitals in Northern Italy over three years.

OBJECTIVES: Genomic surveillance of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) is crucial for virulence, drug-resistance monitoring, and outbreak containment. METHODS: Genomic analysis on 87 KPC-Kp strains isolated from 3 Northern Italy hospitals in 2019-2021 was performed by whole genome sequencing (WGS), to characterize resistome, virulome, and mobilome, and to assess potential associations with phenotype resistance and clinical presentation. Maximum Likelihood and Minimum Spanning Trees were used to determine strain correlations and identify potential transmission clusters. RESULTS: Overall, 15 different STs were found; the predominant ones included ST307 (35, 40.2%), ST512/1519 (15, 17.2%), ST20 (12, 13.8%), and ST101 (7, 8.1%). 33 (37.9%) KPC-Kp strains were noticed to be in five transmission clusters (median number of isolates in each cluster: 5 [3-10]), four of them characterized by intra-hospital transmission. All 87 strains harbored Tn4401a transposon, carrying blaKPC-3 (48, 55.2%), blaKPC-2 (38, 43.7%), and in one case (1.2%) blaKPC-33, the latter gene conferred resistance to ceftazidime/avibactam (CZA). Thirty strains (34.5%) harbored porin mutations; of them, 7 (8.1%) carried multiple Tn4401a copies. These strains were characterized by significantly higher CZA minimum inhibitory concentration compared with strains with no porin mutations or single Tn4401a copy, respectively, even if they did not overcome the resistance breakpoint of 8 ug/mL. Median 2 (IQR:1-2) virulence factors per strain were detected. The lowest number was observed in ST20 compared to the other STs (p<0.001). While ST307 was associated with infection events, a trend associated with colonization events could be observed for ST20. CONCLUSIONS: Integration of genomic, resistance score, and clinical data allowed us to define a relative diversification of KPC-Kp in Northern Italy between 2019 and 2021, characterized by few large transmission chains and rare inter-hospital transmission. Our results also provided initial evidence of correlation between KPC-Kp genomic signatures and higher MIC levels to some antimicrobial agents or colonization/infection status, once again underlining WGS's importance in bacterial surveillance.

The Prevalence of Multidrug-Resistant Escherichia coli in Chennai and Whole Genome Sequence Analysis of Carbapenem-Resistant Escherichia coli ST410.

The current study evaluates antibiotic susceptibility and Extended Spectrum ß-Lactamase (ESBL) production of 557 Escherichia coli isolates obtained from clean catch midstream urine samples using VITEK 2 compact automated microbial identification system. Different classes of drugs were used to determine the Minimum inhibitory concentration (MIC). In our study, 50.45% of isolates were ESBL producers. There is a higher incidence of UTI in females (77.4%) than in males (22.6%). The isolates reveal a high percentage of resistance to antibiotics like nalidixic acid (89.59%), ampicillin (75.76%), ticarcillin (73.43%), cefalotin (67.68%), cefixime (65.17%), ciprofloxacin (58.35%) and ceftriaxone (56.37%). An increased susceptibility pattern was observed for the isolates against drug classes like fosfomycin (98.03%) and nitrofurantoin (91.02%). Among the isolates, 395 (70.91%) were classified as Multidrug-resistant organisms based on the resistance pattern observed against three or more classes of antibiotics. One of the isolates resistant to fluoroquinolones, penicillins, penicillins along with ß-lactamase inhibitor, aminoglycosides, third-generation cephalosporins and carbapenems was subjected to Whole genome sequencing (WGS). WGS data revealed the isolate to be a high-risk clone ST410, which contains antimicrobial-resistance genes (blaTEM-1B, blaCTX-M-15, blaNDM-5, aac(3)-IId, armA, gyrA(p.S83L), gyrA(p.D87N)) conferring resistance to ß-lactam, cephalosporins, carbapenem, aminoglycoside and fluoroquinolone class of antibiotics. The core genome MLST was carried out using BacWGSTdb to assess the global phylogenetic relationship of the genome sequence. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01125-1.

Genome-wide comparative analyses highlights selection signatures underlying saline adaptation in Chilika buffalo.

Chilika, a native buffalo breed of the Eastern coast of India, is mainly distributed around the Chilika brackish water lake connected with the Bay of Bengal Sea. This breed possesses a unique ability to delve deep into the salty water of the lake and stay there to feed on local vegetation of saline nature. Adaptation to salinity is a genetic phenomenon, however, the genetic basis underlying the salinity tolerance is still limited in animals specifically in livestock. The present study explores the genetic evolution that unveils the Chilika buffalo's adaptation to the harsh saline habitat (water and food system). For this study, whole genome resequencing data on 18 Chilika buffalo and for comparison 10 Murrah buffalo of normal habitat were generated. For identification of selection sweeps, intrapopulation and interpopulation statistics were employed. A total of 709, 309, 468, and 354 genes were detected having selection sweeps in Chilika buffalo using the nucleotide diversity (θπ), Tajima's D, nucleotide diversity ratio (θπ-ratio), and FST methods, respectively. Further analysis revealed a total of 23 genes including EXOC6B, VPS8, LYPD1, VPS35, CAMKMT, NCKAP5, COMMD1, MYLK3, B3GNT2 were found to be common by all the methods. Furthermore, functional annotation study of identified genes provided pathways such as MAPK signaling, renin secretion, endocytosis, oxytocin signaling pathway, etc. Gene network analysis enlists hub genes, provide insights into their interactions with each other. In conclusion, this study has highlighted the genetic basis underlying the local adaptive function of Chilika buffalo under saline environment.

Use of Whole Genome Sequencing for Mycobacterium tuberculosis Complex Antimicrobial Susceptibility Testing.

Whole genome sequencing (WGS) is becoming an important diagnostic tool for antimicrobial susceptibility testing of Mycobacterium tuberculosis complex (MTBC) isolates in many countries. WGS protocols usually start with the preparation of a DNA-library: the critical first step in the process. A DNA-library represents the genomic content of a DNA sample and consists of unique short DNA fragments. Although available DNA-library protocols come with manufacturer instructions, details of the entire process, including quality controls, instrument parameters, and run evaluations, often need to be developed and customized by each laboratory to implement WGS technology effectively. Here, we provide a detailed workflow for a DNA-library preparation based on an adapted Illumina protocol optimized for the reduction of reagent costs.

An overview of next generation sequencing strategies and genomics tools used for tuberculosis research.

Tuberculosis (TB) is a grave public health concern and is considered the foremost contributor to human mortality resulting from infectious disease. Due to the stringent clonality and extremely restricted genomic diversity, conventional methods prove inefficient for in-depth exploration of minor genomic variations and the evolutionary dynamics operating in Mycobacterium tuberculosis (M.tb) populations. Until now, the majority of reviews have primarily focused on delineating the application of whole-genome sequencing (WGS) in predicting antibiotic resistant genes, surveillance of drug resistance strains, and M.tb lineage classifications. Despite the growing use of Next Generation Sequencing (NGS) and WGS analysis in tuberculosis (TB) research, there are limited studies that provide a comprehensive summary of its role in studying macroevolution, minor genetic variations, assessing mixed TB infections, and tracking transmission networks at an individual level. This highlights the need for systematic effort to fully explore the potential of WGS and its associated tools in advancing our understanding of TB epidemiology and disease transmission. We delve into the recent bioinformatics pipelines and Next-Generation Sequencing (NGS) strategies that leverage various genetic features and simultaneous exploration of host-pathogen protein expression profile to decipher the genetic heterogeneity and host-pathogen interaction dynamics of the M.tb infections. This review highlights the potential benefits and limitations of NGS and bioinformatics tools and discusses their role in TB detection and epidemiology. Overall, this review could be a valuable resource for researchers and clinicians interested in NGS-based approaches in TB research.

Multi-Allelic Mitochondrial DNA Deletions in an Adult Dog with Chronic Weakness, Exercise Intolerance and Lactic Acidemia.

(1) Background: An adult dog was presented to a board-certified veterinary neurologist for evaluation of chronic weakness, exercise intolerance and lactic acidemia. (2) Methods: A mitochondrial myopathy was diagnosed based on the histological and histochemical phenotype of numerous COX-negative muscle fibers. Whole-genome sequencing established the presence of multiple extended deletions in the mitochondrial DNA (mtDNA), with the highest prevalence between the 1-11 kb positions of the approximately 16 kb mitochondrial chromosome. Such findings are typically suggestive of an underlying nuclear genome variant affecting mitochondrial replication, repair, or metabolism. (3) Results: Numerous variants in the nuclear genome unique to the case were identified in the whole-genome sequence data, and one, the insertion of a DYNLT1 retrogene, whose parent gene is a regulator of the mitochondrial voltage-dependent anion channel (VDAC), was considered a plausible causal variant. (4) Conclusions: Here, we add mitochondrial deletion disorders to the spectrum of myopathies affecting adult dogs.

Evolution of mutations in the ftsI gene leading to amino acid substitutions in PBP3 in Haemophilus influenzae strains under the selective pressure of ampicillin and cefuroxime.

BACKGROUND: Aminopenicillins are recommended agents for non-invasive Haemophilus influenzae infections. One of the mechanisms of resistance to ß-lactams is the alteration of the transpeptidase region of penicillin binding protein 3 (PBP3) which is caused by mutations in the ftsI gene. It was shown that exposure to beta-lactams has a stimulating effect on increase of prevalence of H. influenzae strains with the non-enzymatic mechanism of resistance. OBJECTIVES: The aim of our study was to compare the mutational potential of ampicillin and cefuroxime in H. influenzae strains, determination of minimum inhibitory concentration and the evolution of mutations over time, focusing on amino acid substitutions in PBP3. METHODS: 30 days of serial passaging of strains in liquid broth containing increasing concentrations of ampicillin or cefuroxime was followed by whole-genome sequencing. RESULTS: On average, cefuroxime increased the minimum inhibitory concentration more than ampicillin. The minimum inhibitory concentration was increased by a maximum of 32 fold. Substitutions in the PBP3 started to appear after 15 days of passaging. In PBP3, cefuroxime caused different substitutions than ampicillin. CONCLUSIONS: Our experiment observed differences in mutation selection by ampicillin and cefuroxime. Selection pressure of antibiotics in vitro generated substitutions that do not occur in clinical strains in the Czech Republic.

Foodborne pathogenic bacteria in wild European hedgehogs (Erinaceus europaeus).

BACKGROUND: European hedgehogs (Erinaceus europaeus) are widely distributed across Europe. They may play an important role by spreading zoonotic bacteria in the environment and to humans and animals. The aim of our work was to study the prevalence and characteristics of the most important foodborne bacterial pathogens in wild hedgehogs. RESULTS: Faecal samples from 148 hospitalised wild hedgehogs originating from the Helsinki region in southern Finland were studied. Foodborne pathogens were detected in 60% of the hedgehogs by PCR. Listeria (26%) and STEC (26%) were the most common foodborne pathogens. Salmonella, Yersinia, and Campylobacter were detected in 18%, 16%, and 7% of hedgehogs, respectively. Salmonella and Yersinia were highly susceptible to the tested antimicrobials. Salmonella Enteritidis and Listeria monocytogenes 2a were the most common types found in hedgehogs. All S. Enteritidis belonged to one sequence type (ST11), forming four clusters of closely related isolates. L. monocytogenes was genetically more diverse than Salmonella, belonging to 11 STs. C. jejuni ST45 and ST677, Y. pseudotuberculosis O:1 of ST9 and ST42, and Y. enterocolitica O:9 of ST139 were also found. CONCLUSIONS: Our study shows that wild European hedgehogs should be considered an important source of foodborne pathogens, and appropriate hygiene measures after any contact with hedgehogs and strict biosecurity around farms are therefore important.

Potential clinical applications of advanced genomic analysis in cerebral palsy.

Cerebral palsy (CP) has historically been attributed to acquired insults, but emerging research suggests that genetic variations are also important causes of CP. While microarray and whole-exome sequencing based studies have been the primary methods for establishing new CP-gene relationships and providing a genetic etiology for individual patients, the cause of their condition remains unknown for many patients with CP. Recent advancements in genomic technologies offer additional opportunities to uncover variations in human genomes, transcriptomes, and epigenomes that have previously escaped detection. In this review, we outline the use of these state-of-the-art technologies to address the molecular diagnostic challenges experienced by individuals with CP. We also explore the importance of identifying a molecular etiology whenever possible, given the potential for genomic medicine to provide opportunities to treat patients with CP in new and more precise ways.

Genome analysis of multidrug resistant Enterococcus faecium and Enterococcus faecalis circulating among hospitalized patients in uMgungundlovu District, KwaZulu-Natal, South Africa.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are important pathogens categorized as high-priority bacteria in the Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics published by the World Health Organization. The aim of this study was to determine the risk factors, resistance, virulence, mobilomes associated with multidrug-resistant and clonal lineages of Enterococcus faecium and faecalis circulating among hospitalized patients following the health system in South Africa, using whole genome sequencing (WGS). METHODS: A cross-sectional study was conducted during a two-month periods among hospitalized patients in 2017. Rectal swabs were collected from patients admitted to medical and surgical wards in an urban tertiary hospital, and a rural district hospital in uMgungundlovu district, South Africa. Enterococci were screened for vancomycin resistance on bile esculin azide agar supplemented with 6 mg/L of vancomycin and confirmation of VRE was done using ROSCO kits. Conventional and real-time PCR methods were used to ascertain the presence of VanA, VanB, VanC-2/3 and VanC-1 genes. All six multidrug-resistant Enterococcus faecalis and faecium selected were identified using multiplexed paired-end libraries (2 × 300 bp) with the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) and genome sequencing was done using Illumina MiSeq instrument with 100× coverage at the National Institute of Communicable Diseases Sequencing Core Facility, South Africa. Antibiotic resistance genes, virulence factors, plasmids, integrons and CRISPR were characterized using RAST, ResFinder, VirulenceFinder, PlasmidFinder, PHAST and ISFinder respectively. RESULTS: Sequencing analysis revealed that these strains harbouring numerous resistance genes to glycopeptides (vanC[100%], vex3[100%], vex2[83,33%] and vanG[16,66%]), macrolides, lincosamides, sterptogramine B (ermB[33,32%], Isa[16,66%], emeA[16,66%]) and tetracyclines (tetM[33,32%]) in both district and tertiary hospitals. Multidrug efflux pumps including MATE, MFS and pmrA conferring resistance to several classes of antibiotics were also identified. The main transposable elements observed were in the Tn3 family, specifically Tn1546. Four single sequence types (STs) were identified among E. faecium in the district hospital, namely ST822, ST636, ST97 along with a novel ST assigned ST1386, while one lineage, ST29 was detected in the tertiary hospital. CONCLUSION: The study reveals the genetic diversity and high pathogenicity of multidrug-resistant Enterococcus faecalis and faecium circulating among hospitalized patients. It underlines the necessity to implement routine screening of admitted patients coupled with infection control procedures, antimicrobial stewardship and awareness should be strengthened to prevent and/or contain the carriage and spread of multidrug resistant E. faecium and E. faecalis in hospitals and communities in South Africa.

Pathogenomics analysis of high-risk clone ST147 multidrug-resistant Klebsiella pneumoniae isolated from a patient in Egypt.

BACKGROUND: The emergence of multi-drug-resistant Klebsiella pneumoniae (MDR-KP) represents a serious clinical health concern. Antibiotic resistance and virulence interactions play a significant role in the pathogenesis of K. pneumoniae infections. Therefore, tracking the clinical resistome and virulome through monitoring antibiotic resistance genes (ARG) and virulence factors in the bacterial genome using computational analysis tools is critical for predicting the next epidemic. METHODS: In the current study, one hundred extended spectrum ß-lactamase (ESBL)-producing clinical isolates were collected from Mansoura University Hospital, Egypt, in a six-month period from January to June 2022. One isolate was selected due to the high resistance phenotype, and the genetic features of MDR-KP recovered from hospitalized patient were investigated. Otherwise, the susceptibility to 25 antimicrobials was determined using the DL Antimicrobial Susceptibility Testing (AST) system. Whole genome sequencing (WGS) using Illumina NovaSeq 6000 was employed to provide genomic insights into K. pneumoniae WSF99 clinical isolate. RESULTS: The isolate K. pneumoniae WSF99 was phenotypically resistant to the antibiotics under investigation via antibiotic susceptibility testing. WGS analysis revealed that WSF99 total genome length was 5.7 Mb with an estimated 5,718 protein-coding genes and a G + C content of 56.98 mol%. Additionally, the allelic profile of the WSF99 isolate was allocated to the high-risk clone ST147. Furthermore, diverse antibiotic resistance genes were determined in the genome that explain the high-level resistance phenotypes. Several ß-lactamase genes, including blaCTX-M-15, blaTEM-1, blaTEM-12, blaSHV-11, blaSHV-67, and blaOXA-9, were detected in the WSF99 isolate. Moreover, a single carbapenemase gene, blaNDM-5, was predicted in the genome, positioned within a mobile cassette. In addition, other resistance genes were predicted in the genome including, aac(6')-Ib, aph(3')-VI, sul1, sul2, fosA, aadA, arr-2, qnrS1, tetA and tetC. Four plasmid replicons CoIRNAI, IncFIB(K), IncFIB(pQil), and IncR were predicted in the genome. The draft genome analysis revealed the occurrence of genetic mobile elements positioned around the ARGs, suggesting the ease of dissemination via horizontal gene transfer. CONCLUSIONS: This study reports a comprehensive pathogenomic analysis of MDR-KP isolated from a hospitalized patient. These findings could be relevant for future studies investigating the diversity of antimicrobial resistance and virulence in Egypt.

Comparative genomic analysis of Bacillus atrophaeus HAB-5 reveals genes associated with antimicrobial and plant growth-promoting activities.

Bacillus atrophaeus HAB-5 is a plant growth-promoting rhizobacterium (PGPR) that exhibits several biotechnological traits, such as enhancing plant growth, colonizing the rhizosphere, and engaging in biocontrol activities. In this study, we conducted whole-genome sequencing of B. atrophaeus HAB-5 using the single-molecule real-time (SMRT) sequencing platform by Pacific Biosciences (PacBio; United States), which has a circular chromosome with a total length of 4,083,597 bp and a G + C content of 44.21%. The comparative genomic analysis of B. atrophaeus HAB-5 with other strains, Bacillus amyloliquefaciens DSM7, B. atrophaeus SRCM101359, Bacillus velezensis FZB42, B. velezensis HAB-2, and Bacillus subtilis 168, revealed that these strains share 2,465 CDSs, while 599 CDSs are exclusive to the B. atrophaeus HAB-5 strain. Many gene clusters in the B. atrophaeus HAB-5 genome are associated with the production of antimicrobial lipopeptides and polypeptides. These gene clusters comprise distinct enzymes that encode three NRPs, two Transat-Pks, one terpene, one lanthipeptide, one T3PKS, one Ripp, and one thiopeptide. In addition to the likely IAA-producing genes (trpA, trpB, trpC, trpD, trpE, trpS, ywkB, miaA, and nadE), there are probable genes that produce volatile chemicals (acoA, acoB, acoR, acuB, and acuC). Moreover, HAB-5 contained genes linked to iron transportation (fbpA, fetB, feuC, feuB, feuA, and fecD), sulfur metabolism (cysC, sat, cysK, cysS, and sulP), phosphorus solubilization (ispH, pstA, pstC, pstS, pstB, gltP, and phoH), and nitrogen fixation (nif3-like, gltP, gltX, glnR, glnA, nadR, nirB, nirD, nasD, narl, narH, narJ, and nark). In conclusion, this study provides a comprehensive genomic analysis of B. atrophaeus HAB-5, pinpointing the genes and genomic regions linked to the antimicrobial properties of the strain. These findings advance our knowledge of the genetic basis of the antimicrobial properties of B. atrophaeus and imply that HAB-5 may employ a variety of commercial biopesticides and biofertilizers as a substitute strategy to increase agricultural output and manage a variety of plant diseases.

Post-implantation analysis of genomic variations in the progeny from developing fetus to birth.

The analysis of genomic variations in offspring after implantation has been infrequently studied. In this study, we aim to investigate the extent of de novo mutations in humans from developing fetus to birth. Using high-depth whole-genome sequencing, 443 parent-offspring trios were studied to compare the results of de novo mutations (DNMs) between different groups. The focus was on fetuses and newborns, with DNA samples obtained from the families' blood and the aspirated embryonic tissues subjected to deep sequencing. It was observed that the average number of total DNMs in the newborns group was 56.26 (54.17-58.35), which appeared to be lower than that the multifetal reduction group, which was 76.05 (69.70-82.40) (F = 2.42, P = 0.12). However, after adjusting for parental age and maternal pre-pregnancy body mass index (BMI), significant differences were found between the two groups. The analysis was further divided into single nucleotide variants (SNVs) and insertion/deletion of a small number of bases (indels), and it was discovered that the average number of de novo SNVs associated with the multifetal reduction group and the newborn group was 49.89 (45.59-54.20) and 51.09 (49.22-52.96), respectively. No significant differences were noted between the groups (F = 1.01, P = 0.32). However, a significant difference was observed for de novo indels, with a higher average number found in the multifetal reduction group compared to the newborn group (F = 194.17, P < 0.001). The average number of de novo indels among the multifetal reduction group and the newborn group was 26.26 (23.27-29.05) and 5.17 (4.82-5.52), respectively. To conclude, it has been observed that the quantity of de novo indels in the newborns experiences a significant decrease when compared to that in the aspirated embryonic tissues (7-9 weeks). This phenomenon is evident across all genomic regions, highlighting the adverse effects of de novo indels on the fetus and emphasizing the significance of embryonic implantation and intrauterine growth in human genetic selection mechanisms.

Surveillance of vancomycin-resistant enterococci reveals shift in dominating clusters from vanA to vanB Enterococcus faecium clusters, Denmark, 2015 to 2022.

BackgroundVancomycin-resistant enterococci (VRE) are increasing in Denmark and Europe. Linezolid and vancomycin-resistant enterococci (LVRE) are of concern, as treatment options are limited. Vancomycin-variable enterococci (VVE) harbour the vanA gene complex but are phenotypically vancomycin-susceptible.AimThe aim was to describe clonal shifts for VRE and VVE in Denmark between 2015 and 2022 and to investigate genotypic linezolid resistance among the VRE and VVE.MethodsFrom 2015 to 2022, 4,090 Danish clinical VRE and VVE isolates were whole genome sequenced. We extracted vancomycin resistance genes and sequence types (STs) from the sequencing data and performed core genome multilocus sequence typing (cgMLST) analysis for Enterococcus faecium. All isolates were tested for the presence of mutations or genes encoding linezolid resistance.ResultsIn total 99% of the VRE and VVE isolates were E. faecium. From 2015 through 2019, 91.1% of the VRE and VVE were vanA E. faecium. During 2020, to the number of vanB E. faecium increased to 254 of 509 VRE and VVE isolates. Between 2015 and 2022, seven E. faecium clusters dominated: ST80-CT14 vanA, ST117-CT24 vanA, ST203-CT859 vanA, ST1421-CT1134 vanA (VVE cluster), ST80-CT1064 vanA/vanB, ST117-CT36 vanB and ST80-CT2406 vanB. We detected 35 linezolid vancomycin-resistant E. faecium and eight linezolid-resistant VVEfm.ConclusionFrom 2015 to 2022, the numbers of VRE and VVE increased. The spread of the VVE cluster ST1421-CT1134 vanA E. faecium in Denmark is a concern, especially since VVE diagnostics are challenging. The finding of LVRE, although in small numbers, ia also a concern, as treatment options are limited.

Development and application of a whole genome amplicon sequencing method for infectious salmon anemia virus (ISAV).

Infectious salmon anemia (ISA) is an infectious disease primarily affecting farmed Atlantic salmon, Salmo salar, which is caused by the ISA virus (ISAV). ISAV belongs to the Orthomyxoviridae family. The disease is a serious condition resulting in reduced fish welfare and high mortality. In this study, we designed an amplicon-based sequencing protocol for whole genome sequencing of ISAV. The method consists of 80 ISAV-specific primers that cover 92% of the virus genome and was designed to be used on an Illumina MiSeq platform. The sequencing accuracy was investigated by comparing sequences with previously published Sanger sequences. The sequences obtained were nearly identical to those obtained by Sanger sequencing, thus demonstrating that sequences produced by this amplicon sequencing protocol had an acceptable accuracy. The amplicon-based sequencing method was used to obtain the whole genome sequence of 12 different ISAV isolates from a small local epidemic in the northern part of Norway. Analysis of the whole genome sequences revealed that segment reassortment took place between some of the isolates and could identify which segments that had been reassorted.

A Strategy for Studying Epigenetic Diversity in Natural Populations: Proof of Concept in Poplar and Oak.

These last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulphite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions (DMRs) as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, considering the structural complexity of the genomes with their size and their content in unique as coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a representative subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by Sequencing Capture Bisulphite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea.

The vaginal microbial signatures of preterm birth woman.

To explore the differences of vaginal microbes in women with preterm birth (PTB), and to construct prediction model. We searched for articles related to vaginal microbiology in preterm women and obtained four 16S rRNA-sequence datasets. We analyzed that for species diversity and differences, and constructed a random forest model with 20 differential genera. We introduce an independent whole genome-sequencing (WGS) data for validation. In addition, we collected vaginal and cervical swabs from 33 pregnant women who delivered spontaneously full-term and preterm infants, performed WGS in our lab to further validate the model. Compared to term birth (TB) samples, PTB women vagina were characterized by a decrease in Firmicutes, Lactobacillus, and an increase in diversity accompanied by the colonization of pathogenic bacteria such as Gardnerella, Atopobium and Prevotella. Twenty genus markers, including Lactobacillus, Prevotella, Streptococcus, and Gardnerella performed well in predicting PTB, with study-to-study transfer validation and LODO validation, different gestation validation showing good results, and in two independent cohorts (external WGS cohorts and woman samples WGS cohorts) in which the accuracy was maintained. PTB women have unique vaginal microbiota characteristics. A predictive model of PTB was constructed and its value validated from multiple perspectives.

Known pathogenic gene variants and new candidates detected in sudden unexpected infant death using whole genome sequencing.

The purpose of this study is to gain insights into potential genetic factors contributing to the infant's vulnerability to Sudden Unexpected Infant Death (SUID). Whole Genome Sequencing (WGS) was performed on 144 infants that succumbed to SUID, and 573 healthy adults. Variants were filtered by gnomAD allele frequencies and predictions of functional consequences. Variants of interest were identified in 88 genes, in 64.6% of our cohort. Seventy-three of these have been previously associated with SIDS/SUID/SUDP. Forty-three can be characterized as cardiac genes and are related to cardiomyopathies, arrhythmias, and other conditions. Variants in 22 genes were associated with neurologic functions. Variants were also found in 13 genes reported to be pathogenic for various systemic disorders and in two genes associated with immunological function. Variants in eight genes are implicated in the response to hypoxia and the regulation of reactive oxygen species (ROS) and have not been previously described in SIDS/SUID/SUDP. Seventy-two infants met the triple risk hypothesis criteria. Our study confirms and further expands the list of genetic variants associated with SUID. The abundance of genes associated with heart disease and the discovery of variants associated with the redox metabolism have important mechanistic implications for the pathophysiology of SUID.

Methods of Detection and Mechanisms of Origin of Complex Structural Genome Variations.

Based on classical karyotyping, structural genome variations (SVs) have generally been considered to be either "simple" (with one or two breakpoints) or "complex" (with more than two breakpoints). Studying the breakpoints of SVs at nucleotide resolution revealed additional, subtle structural variations, such that even "simple" SVs turned out to be "complex." Genome-wide sequencing methods, such as fosmid and paired-end mapping, short-read and long-read whole genome sequencing, and single-molecule optical mapping, also indicated that the number of SVs per individual was considerably larger than expected from karyotyping and high-resolution chromosomal array-based studies. Interestingly, SVs were detected in studies of cohorts of individuals without clinical phenotypes. The common denominator of all SVs appears to be a failure to accurately repair DNA double-strand breaks (DSBs) or to halt cell cycle progression if DSBs persist. This review discusses the various DSB response mechanisms during the mitotic cell cycle and during meiosis and their regulation. Emphasis is given to the molecular mechanisms involved in the formation of translocations, deletions, duplications, and inversions during or shortly after meiosis I. Recently, CRISPR-Cas9 studies have provided unexpected insights into the formation of translocations and chromothripsis by both breakage-fusion-bridge and micronucleus-dependent mechanisms.