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The global spread and continued evolution of SARS-CoV-2 has driven an unprecedented surge in viral genomic surveillance. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine laboratory processes and results. This challenge will increase with the expanding global production of sequences across a variety of laboratories for epidemiological and clinical interpretation, as well as for genomic surveillance of emerging diseases in future outbreaks. We present SDSI + AmpSeq, an approach that uses 96 synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination throughout the sequencing workflow. We apply SDSIs to the ARTIC Consortium's amplicon design, demonstrate their utility and efficiency in a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases and validate them across 6,676 diagnostic samples at multiple laboratories. We establish that SDSI + AmpSeq provides increased confidence in genomic data by detecting and correcting for relatively common, yet previously unobserved modes of error, including spillover and sample swaps, without impacting genome recovery., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected the whole world, including Odisha, a state in eastern India. Many people have migrated to the state from different countries as well as other states during this SARS-CoV-2 pandemic. The aim of this study was to analyse the receptor-binding domain (RBD) sequence of the spike protein from isolates collected from throat swab samples of COVID-19-positive patients and further to assess the RBD affinity for angiotensin-converting enzyme 2 (ACE2) of different species, including humans., Methods: Whole-genome sequencing for 35 clinical SARS-CoV-2 isolates from COVID-19-positive patients was performed by ARTIC amplicon-based sequencing. Sequence analysis and phylogenetic analysis were performed for the spike region and the RBD region of all isolates. The interaction between the RBD and ACE2 of five different species was also analysed., Results: The spike region of 32 isolates showed one or multiple alterations in nucleotide bases in comparison with the Wuhan reference strain. One of the identified mutations, at position 1204 (Ref A, RMRC 22 C), in the RBD coding region of the spike protein showed stronger binding affinity for human ACE2. Furthermore, RBDs of all the Indian isolates showed binding affinity for ACE2 of different species., Conclusion: As mutant RBD showed stronger interaction with human ACE2, it could potentially result in higher infectivity. The binding affinity of the RBDs for ACE2 of all five species studied suggests that the virus can infect a wide variety of animals, which could also act as natural reservoir for SARS-CoV-2., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Norovirus, a major cause of gastroenteritis in people of all ages worldwide, was first reported in South Korea in 1999. The most common causal agents of pediatric acute gastroenteritis are norovirus and rotavirus. While vaccination has reduced the pediatric rotavirus infection rate, norovirus vaccines have not been developed. Therefore, prediction and prevention of norovirus are very important. Norovirus is divided into genogroups GI-GVII, with GII.4 being the most prevalent. However, in 2012-2013, GII.17 showed a higher incidence than GII.4 and a novel variant, GII.P17-GII.17, appeared. In this study, 204 stool samples collected in 2013-2014 were screened by reverse transcriptase-polymerase chain reaction; 11 GI (5.39%) and 45 GII (22.06%) noroviruses were identified. GI.4, GI.5, GII.4, GII.6 and GII.17 were detected. The whole genomes of the three norovirus GII.17 were sequenced. The whole genome of GII.17 consists of three open reading frames of 5109, 1623 and 780 bp. Compared with 20 GII.17 strains isolated in other countries, we observed numerous changes in the protruding P2 domain of VP1 in the Korean GII.17 viruses. Our study provided genome information that might aid in epidemic prevention, epidemiology studies and vaccine development.

Background: Human Phenotype Ontology (HPO) has risen as a useful tool for precision medicine by providing a standardized vocabulary of phenotypic abnormalities to describe presentations of human pathologies; however, there have been relatively few reports combining whole genome sequencing (WGS) and HPO, especially in the context of structural variants., Methods: We illustrate an integrative analysis of WGS and HPO using an extended pedigree, which involves Prader-Willi Syndrome (PWS), hereditary hemochromatosis (HH), and dysautonomia-like symptoms. A comprehensive WGS pipeline was used to ensure reliable detection of genomic variants. Beyond variant filtering, we pursued phenotypic prioritization of candidate genes using Phenolyzer., Results: Regarding PWS, WGS confirmed a 5.5 Mb de novo deletion of the parental allele at 15q11.2 to 15q13.1. Phenolyzer successfully returned the diagnosis of PWS, and pinpointed clinically relevant genes in the deletion. Further, Phenolyzer revealed how each of the genes is linked with the phenotypes represented by HPO terms. For HH, WGS identified a known disease variant (p.C282Y) in HFE of an affected female. Analysis of HPO terms alone fails to provide a correct diagnosis, but Phenolyzer successfully revealed the phenotype-genotype relationship using a disease-centric approach. Finally, Phenolyzer also revealed the complexity behind dysautonomia-like symptoms, and seven variants that might be associated with the phenotypes were identified by manual filtering based on a dominant inheritance model., Conclusions: The integration of WGS and HPO can inform comprehensive molecular diagnosis for patients, eliminate false positives and reveal novel insights into undiagnosed diseases. Due to extreme heterogeneity and insufficient knowledge of human diseases, it is also important that phenotypic and genomic data are standardized and shared simultaneously.

Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ∼50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the '5mCAdpBS-Seq' workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (∼15%) compared to mncfDNA and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment was correlated with expressed genes of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.

In this study, a novel mitovirus, tentatively designated as "Alternaria alternata mitovirus 2" (AaMV2), was isolated from the fungus Alternaria alternata f. sp. mali causing apple leaf blotch disease. The complete genome of AaMV2 is 3,157 nucleotides in length, with an A+U content of 68.10%. The genome has a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) protein with a molecular mass of 98.10 kDa. BLAST analysis revealed that AaMV2 has the highest sequence identity to Leptosphaeria biglobosa mitovirus 6, with 79.76% and 82.86% identity at the amino acid and nucleotide level, respectively. Phylogenetic analysis suggested that AaMV2 is a new member of the genus Duamitovirus within the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus in A. alternata. [ABSTRACT FROM AUTHOR]

Background: West Nile Virus (WNV) is a zoonotic arbovirus worldwide spread. Seasonal WNV outbreaks occur in the Mediterranean basin since the late 1990's with ever-increasing incidence. In Southern Spain WNV is endemic, as disease foci - caused by WNV lineage 1 (WNV-L1) strains - occur every year. On the contrary, WNV-L2 is the dominant lineage in Europe, so most European WNV sequences available belong to this lineage, WNV-L1 sequences being still scarce. Methods: To fill this gap, this study reports the genetic characterisation of 27 newly described WNV-L1 strains, involved in outbreaks affecting wild birds and horses during the last decade in South-Western Spain. Results: All strains except one belong to the Western Mediterranean-1 sub-cluster (WMed-1), related phylogenetically to Italian, French, Portuguese, Moroccan and, remarkably, Senegalese strains. This sub-cluster persisted, spread and evolved into three distinguishable WMed-1 phylogenetic groups that co-circulated, notably, in the same province (Cádiz). They displayed different behaviours: from long-term persistence and rapid spread to neighbouring regions within Spain, to long-distance spread to different countries, including transcontinental spread to Africa. Among the different introductions of WNV in Spain revealed in this study, some of them succeeded to get established, some extinguished from the territory shortly afterwards. Furthermore, Spain's southernmost province, Cádiz, constitutes a hotspot for virus incursion. Conclusion: Southern Spain seems a likely scenario for emergence of exotic pathogens of African origin. Therefore, circulation of diverse WNV-L1 variants in Spain prompts for an extensive surveillance under a One Health approach. [ABSTRACT FROM AUTHOR]

The Philippines is a high-incidence country for tuberculosis, with the increasing prevalence of multi- (MDR-TB) and extensively-drug (XDR-TB) resistant Mycobacterium tuberculosis strains posing difficulties to disease control. Understanding the genetic diversity of circulating strains can provide insights into underlying drug resistance mutations and transmission dynamics, thereby assisting the design of diagnostic tools, including those using next generation sequencing (NGS) platforms. By analysing genome sequencing data of 732 isolates from Philippines drug-resistance survey collections spanning from 2011 to 2019, we found that the majority belonged to lineages L1 (531/732; 72.5%) and L4 (European-American; n = 174; 23.8%), with the Manila strain (L1.2.1.2.1) being the most prominent (475/531). Approximately two-thirds of isolates were found to be at least MDR-TB (483/732; 66.0%), and potential XDR-TB genotypic resistance was observed (3/732; 0.4%), highlighting an emerging problem in the country. Genotypic resistance was highly concordant with laboratory drug susceptibility testing. By finding isolates with (near-)identical genomic variation, five major clusters containing a total of 114 isolates were identified: all containing either L1 or L4 isolates with at least MDR-TB resistance and spanning multiple years of collection. Closer inspection of clusters revealed transmission in prisons, some involving isolates with XDR-TB, and mutations linked to third-line drug bedaquiline. We have also identified previously unreported mutations linked to resistance for isoniazid, rifampicin, ethambutol, and fluoroquinolones. Overall, this study provides important insights into the genetic diversity, transmission and circulating drug resistance mutations of M. tuberculosis in the Philippines, thereby informing clinical and surveillance decision-making, which is increasingly using NGS platforms. [ABSTRACT FROM AUTHOR]

Since the start of the mandatory nationwide bovine viral diarrhea (BVD) eradication program in Germany in 2011, the number of persistently infected (PI) animals has decreased considerably, resulting in a continuous decrease in seroprevalence. The increasingly BVD-naive cattle population could facilitate spillover infections with non-BVDV ruminant pestiviruses. Here, we report two cases in which novel pestiviruses were isolated from cattle; in both cases, the whole genome sequence showed the highest level of identity to strain "Pestivirus reindeer-1". Both novel viruses gave positive results in BVDV diagnostic test systems, confirming that cross-reactivity is an important issue in pestivirus diagnostics. In the first case, the pestivirus was probably transmitted from sheep kept with the affected cattle, suggesting that the co-housing of small ruminants and cattle is a risk factor. The source of infection could not be determined in the second case. The occurrence of these two cases in independent cattle holdings within a relatively short time frame suggests that it would be useful to determine the presence of pestiviruses in small ruminants or even wild ruminants to better assess risk factors, especially for BVDV-free populations. [ABSTRACT FROM AUTHOR]

Astroviruses are single-stranded, positive-sense RNA viruses capable of infecting humans as well as a wide range of mammalian and avian species, with a length of approximately 6.6–7.7 kb. In this study, 139 goat fecal samples collected from the Guangxi province were used for the RT-PCR detection, and two of these were positive for goat astrovirus, with a positivity rate of 1.44% (2/139). The complete genome sequence of an astrovirus strain and the partial genome sequence of a strain astrovirus, named GX WZ 2023 and GX HC 2023, were amplified and sequenced, and their sequence lengths were 6284 nt and 6213 nt, respectively. Among them, the capsid protein of goat astrovirus GX HC 2023 showed the highest amino acid identity of 95.9% with ovine astrovirus GX, which belonged to the MAstV-2 genotype. However, the closest relative of the GX WZ 2023 strain was found to be the caprine astrovirus Sichuan, with a nucleotide sequence identity of 76.8%. The ORF1ab nonstructural protein of this strain showed the highest amino acid identities of 89.2 and 95.8% with the ovine astrovirus S5.1 and caprine astrovirus G5.1 strains, respectively. However, its ORF2 capsid protein has 68.4% amino acid identity with the bovine astrovirus (BAstV) 16 2021 CHN strain and only 21.9–64% amino acid identity with all available strains of goat astrovirus. The GX WZ 2023 strain was recombined with the Chinese (BAstV 16 2021 CHN) and Japanese bovine strains (BAstV JPN 2015) in the ORF2 region. Therefore, the goat astrovirus GX WZ 2023 is proposed as a new member of the family goat astroviridae based on the species classification criteria of the International Committee on Taxonomy of Viruses. These findings enhance our understanding of the prevalence and genetic evolution of goat astrovirus and provide a scientific basis for future studies of these viruses in other animals. [ABSTRACT FROM AUTHOR]

Microorganisms, genetically modified or not, may be used in the food chain either as active agents, biomasses or as production organisms of substances of interest. The placement of such microorganisms or their derived substances/products in the European market may be subject to a premarket authorisation process. The authorisation process requires a risk assessment in order to establish the safety and/or the efficacy of the microorganism(s) when used in the food chain as such, as biomasses or as production strains. This includes a full molecular characterisation of the microorganism(s) under assessment. For certain regulated products, the use of whole genome sequence (WGS) data of the microorganism is established as a requirement for the risk assessment. In this regard, data obtained from WGS analysis can provide information on the unambiguous taxonomic identification of the strains, on the presence of genes of concern (e.g. those encoding virulence factors, resistance to antimicrobials of clinical relevance for humans and animals, production of harmful metabolites or of clinically relevant antimicrobials) and on the characterisation of genetic modification(s) (where relevant). This document provides recommendations to applicants on how to describe and report the results of WGS analyses in the context of an application for market authorisation of a regulated product. Indications are given on how to perform genome sequencing and the quality criteria/thresholds that should be reached, as well as the data and relevant information that need to be reported, if required. This updated document replaces the EFSA 2021 Statement and reflects the current knowledge in technologies and methodologies to be used to generate and analyse WGS data for the risk assessment of microorganisms. [ABSTRACT FROM AUTHOR]

Background: The omicron variants have been classified as variants of concern by the world health organization due to their initial emergence with a notable mutations that affect on the transmissibility, immune escape and virulence of the SARS-CoV-2 virus. We conducted a cross-sectional analysis study to detect the most common strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Egypt during the 4th wave of the COVID-19 pandemic and used the next genaration sequencing to characterize the SARS CoV-2 omicron variant genome and find the significant mutation influencing the virus's virulence, immune evasion, and transmissibility. Material and methods: This study started during the 4th wave of the COVID-19 pandemic by collecting nasopharyngeal swabs (n = 39) from individuals having COVID-19 symptoms. SARSCoV-2 was molecularly identified using reverse transcription PCR (RT-PCR). For every sample, whole genome sequencing (WGS) was carried out using next generation sequencing Illumina Iseq100 system. Clade assignment, Pangolin lineages, phylogenetic placement, and mutation calling were all carried out using Nextclade tool V3.4.0, Coronavirus Typing Tool V 1.25 and CoVsurver mutation application. Result: Sequencing data analysis proved that all the involved samples in the study belong to the Omicron variant with different clade and Pangolin lineage, the most common strain in Egypt during the 4th wave of the COVID-19 pandemic was Omicron clade 22B and BA.5.2 Pango lineage. Mutation analysis showed that the S gene of the omicron variant accumulated with significant mutations that affect viral immune escape. Conclusion: The omicron variant in Egypt during the 4th wave of the pandemic showed a high ability to escape from the immune system because of newly mutation on the antibodies recognized sites of the S gene, so it is very important to perform continuous monitoring for omicron genome mutations using next-generation sequencing to help the scientific community in the development of appropriate vaccine for this variant. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming, and ubiquitous bacterium harboring plasmids encoding a variety of proteins with insecticidal activity, but also with activity against nematodes. The aim of this work was to perform the genome sequencing and analysis of a native Bt strain showing bipyramidal parasporal crystals and designated V-CO3.3, which was isolated from the dust of a grain storehouse in Córdoba (Spain). Its genome comprised 99 high-quality assembled contigs accounting for a total size of 5.2 Mb and 35.1% G + C. Phylogenetic analyses suggested that this strain should be renamed as Bacillus cereus s.s. biovar Thuringiensis. Gene annotation revealed a total of 5495 genes, among which, 1 was identified as encoding a Cry5Ba homolog protein with well-documented toxicity against nematodes. These results suggest that this Bt strain has interesting potential for nematode biocontrol. Dataset: The BioSample data have been submitted under accession number SAMN41014059. The whole-genome sequences have been submitted to the NCBI with BioProject number PRJNA1102171. The assembled genome sequences are available under accession number JBCGZW000000000. The (Illumina) raw reads have been submitted to Sequence Read Archives (SRA) under accession number SRR28927673. The V-CO-3.3 strain has been stored in the Spanish Type Culture Collection (CECT) at the University of Valencia (Spain) under accession number CECT 31068. Dataset License: CC-BY-NC [ABSTRACT FROM AUTHOR]

Carbapenemase-producing Pseudomonas aeruginosa strains present a specific geographical distribution regarding the type of carbapenemase-encoding genes that they harbor. For more than twenty years, VIM-type enzymes were the only major carbapenemases that were detected among P. aeruginosa isolates in Greece until the emergence of NDM-1-encoding P. aeruginosa in early 2023. In the present study, we present the rapid reversal of the carbapenemase-producing P. aeruginosa epidemiology from blaVIM- to blaNDM-harbouring isolates that occurred in our hospital since then. Between January 2023 and February 2024, 139 isolates tested positive for carbapenemase production with the NG-Test CARBA 5 immunochromatographic assay. Eight isolates were processed with the Hybrispot antimicrobial resistance direct flow chip molecular assay, and the first NDM-producing isolate was further analyzed through whole genome sequencing and bioinformatics analysis. Multiple resistance genes were detected by molecular techniques in accordance with the extensively drug-resistant phenotype. The isolate that was subjected to whole-genome sequencing belonged to the P. aeruginosa high-risk clone ST308, and the blaNDM was located in the chromosome in accordance with previously reported data. During the study period, NDM-producing isolates were increasingly detected, and only five months after their emergence, they overcame VIM producers. Our results indicate the potential of this new clone to spread rapidly and predominate within healthcare institutions, further restricting the already limited treatment options. [ABSTRACT FROM AUTHOR]

The classification and identification of species in Populus has remained a formidable challenge due to widespread interspecies hybridization. The complete chloroplast genome of Populus maximowiczii was obtained by Illumina high-throughput sequencing technology, with a typical quadripartite structure and 37.0% GC content. The chloroplast genome of P. maximowiczii was 156,892 in length, including a large single-copy region (LSC: 84,988 bp), a small single-copy region (SSC: 16,630 bp), and a pair of inverted repeats (IRs: each 27,637 bp in length). A total of 131 genes were annotated, including 86 protein-coding genes, 37 tRNAs, and 8 rRNAs. The phylogenetic analysis indicated that 43 species belonging to Populus were classified into monophyly, with P. cathayana being the closest relatives to P. maximowiczii. In conclusion, this study provides valuable insights into understanding the phylogeny of Populus. [ABSTRACT FROM AUTHOR]

Currently, the phylogenetic relationships of Tragopogon pratensis Linnaeus (1753) remain unclear. This study presents the first report on the complete chloroplast genome of T. pratensis, which is a quadripartite structure with a length of 153,002 bp and containing a large single copy (LSC, 84,225 bp) region, a small single copy (SSC, 18,407 bp) region, a pair of inverted repeats (IR, 25,185 bp) regions. A total of 134 genes are identified, including 87 protein-coding genes, 8 rRNA genes, 37 tRNA genes, and 2 pseudogenes. Phylogenetic analysis revealed that T. pratensis is most closely related to T. dubius and that Tragopogon is a monophyletic genus. [ABSTRACT FROM AUTHOR]

Avian metapneumovirus (aMPV) poses a significant threat to the poultry industry worldwide, primarily affecting turkeys and chickens. The recent detection of aMPV-A and -B subtypes in the United States marks a significant shift after a prolonged period free of aMPV following the eradication of the previously circulating subtype C. Hence, the demand for molecular diagnostic tests for aMPV has arisen due to their limited availability in the US market. In this study, we present the molecular characterization based on the complete genome sequence of aMPV subtype A, which was detected in the US for the first time. Four RT-qPCR positive samples were subjected to next-generation sequencing analysis, resulting in the assembly of one complete and one near-complete genome sequences. Phylogenetic analysis revealed that the isolated strains clustered within the aMPV-A subtype and were most closely related to recent Mexican strains. A detailed amino acid analysis identified unique mutations in the G gene of the US isolates compared to Mexican strains. Additionally, we compared the performance, cross-reactivity, and limit of detection of our revised aMPV subtype-specific RT-qPCR test with two commercial kits, demonstrating similar detection and subtyping capabilities. These findings highlight the importance of accurate diagnostic methods for disease management in the poultry industry, provide valuable insights into the epidemiology of aMPV, and underscore the need for continued vigilance and surveillance to mitigate its impact on poultry production. [ABSTRACT FROM AUTHOR]

In order to resolve the key genes for weed control by Trichoderma polysporum at the genomic level, we extracted the genomic DNA and sequenced the whole genome of T. polysporum strain HZ-31 on the Illumina Hiseq platform. The raw data was cleaned up using Trimmomatic and checked for quality using FastQC. The sequencing data was assembled using SPAdes, and GeneMark was used to perform gene prediction on the assembly results. The results showed that the genome size of T. polysporum HZ-31 was 39,325,746 bp, with 48% GC content, and the number of genes encoded was 11,998. A total of 148 tRNAs and 45 rRNAs were predicted. A total of 782 genes were annotated in the Carbohydrase Database, 757 genes were annotated to the Pathogen-Host Interaction Database, and 67 gene clusters were identified. In addition, 1023 genes were predicted to be signal peptide proteins. The annotation and functional analysis of the whole genome sequence of T. polymorpha HZ-31 provide a basis for the in-depth study of the molecular mechanism of its herbicidal action and more effective utilization for weed control. [ABSTRACT FROM AUTHOR]

Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 virus was detected in the South American sea lions found dead in Santa Catarina, Brazil, in October 2023. Whole genome sequencing and comparative phylogenetic analysis were conducted to investigate the origin, genetic diversity, and zoonotic potentials of the H5N1 viruses. The H5N1 viruses belonged to the genotype B3.2 of clade 2.3.4.4b H5N1 virus, which was identified in North America and disseminated to South America. They have acquired new amino acid substitutions related to mammalian host affinity. Our study provides insights into the genetic landscape of HPAI H5N1 viruses in Brazil, highlighting the continuous evolutionary processes contributing to their possible adaptation to mammalian hosts. [ABSTRACT FROM AUTHOR]

The zoonotic Shiga toxin-producing Escherichia coli (STEC) group is unanimously regarded as exceptionally hazardous for humans. This study aimed to provide a genomic perspective on the STEC recovered sporadically from humans and have a foundation of internationally comparable data. Fifty clinical STEC isolates, representing the culture-confirmed infections reported by the STEC Reference Laboratory between 2016 and 2023, were subjected to whole-genome sequencing (WGS) analysis and sequences were interpreted using both commercial and public free bioinformatics tools. The WGS analysis revealed a genetically diverse population of STEC dominated by non-O157 serogroups commonly reported in human STEC infections in the European Union. The O26:H11 strains of ST21 lineage played a major role in the clinical disease resulting in hospitalisation and cases of paediatric HUS in Romania surpassing the O157:H7 strains. The latter were all clade 7 and mostly ST1804. Notably, among the Romanian isolates was a stx2a-harbouring cryptic clade I strain associated with a HUS case, stx2f- and stx2e-positive strains, and hybrid strains displaying a mixture of intestinal and extraintestinal virulence genes were found. As a clearer picture emerges of the STEC strains responsible for infections in Romania, further surveillance efforts are needed to uncover their prevalence, sources, and reservoirs. [ABSTRACT FROM AUTHOR]

Age-associated changes in DNA methylation have been characterized across various animals, but not yet in amphibians, which are of particular interest because they include widely studied model organisms. In this study, we present clear evidence that the aquatic vertebrate species Xenopus tropicalis displays patterns of age-associated changes in DNA methylation. We have generated whole-genome bisulfite sequencing (WGBS) profiles from skin samples of nine frogs representing young, mature, and old adults and characterized the gene- and chromosome-scale DNA methylation changes with age. Many of the methylation features and changes we observe are consistent with what is known in mammalian species, suggesting that the mechanism of age-related changes is conserved. Moreover, we selected a few thousand age-associated CpG sites to build an assay based on targeted DNA methylation analysis (TBSseq) to expand our findings in future studies involving larger cohorts of individuals. Preliminary results of a pilot TBSeq experiment recapitulate the findings obtained with WGBS setting the basis for the development of an epigenetic clock assay. The results of this study will allow us to leverage the unique resources available for Xenopus to study how DNA methylation relates to other hallmarks of ageing.

Objective: Rare disease Background: Flavonifractor plautii belongs to the clostridium family, which can lead to local infections as well as the bloodstream infections. Flavonifractor plautii caused infection is rarely few in the clinic. To understand better Flavonifractor plautii, we investigated the drug sensitivity and perform genome sequencing of Flavonifractor plautii isolated from blood samples in China and explored the drug resistance and pathogenic mechanism of the bacteria. Case report: The Epsilometer test method was used to detect the sensitivity of flavonoid bacteria to antimicrobial agents. PacBio sequencing technology was employed to sequence the whole genome of Flavonifractor plautii, and gene prediction and functional annotation were also analyzed. Flavonifractor plautii displayed sensitivity to most drugs but resistance to fluoroquinolones and tetracycline, potentially mediated by tet (W/N/W). The total genome size of Flavonifractor plautii was 4,573,303 bp, and the GC content was 59.78%. Genome prediction identified 4,506 open reading frames, including 9 ribosomal RNAs and 66 transfer RNAs. It was detected that the main virulence factor-coding genes of the bacteria were the capsule, polar flagella and FbpABC, which may be associated with bacterial movement, adhesion, and biofilm formation. Conclusions: The results of whole-genome sequencing could provide relevant information about the drug resistance mechanism and pathogenic mechanism of bacteria and offer a basis for clinical diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Many bacterial pathogens, including the human exclusive pathogen Salmonella Typhi, express capsular polysaccharides as a crucial virulence factor. Here, through S. Typhi whole genome sequence analyses and functional studies, we found a list of single point mutations that make S. Typhi hypervirulent. We discovered a single point mutation in the Vi biosynthesis enzymes that control Vi polymerization or acetylation is enough to result in different capsule variants of S. Typhi. All variant strains are pathogenic, but the hyper Vi capsule variants are particularly hypervirulent, as demonstrated by the high morbidity and mortality rates observed in infected mice. The hypo Vi capsule variants have primarily been identified in Africa, whereas the hyper Vi capsule variants are distributed worldwide. Collectively, these studies increase awareness about the existence of different capsule variants of S. Typhi, establish a solid foundation for numerous future studies on S. Typhi capsule variants, and offer valuable insights into strategies to combat capsulated bacteria. Here, Lee and Song describe missense mutations in the viaB locus in clinical isolates of Salmonella Typhi that result in hypo- and hyper-virulent phenotypes due to changes in the capsule. [ABSTRACT FROM AUTHOR]

[Display omitted] • Whole-genome sequencing and comparative genomic analysis were performed on Cryptosporidium xiaoi. • Cryptosporidium xiaoi and the bovine-specific C. bovis have 96% genomic identity and undergo similar genome reduction. • The species-specific and divergent genes between C. xiaoi and C. bovis may contribute to their different host preferences. • These genes may be targets for in-depth study of Cryptosporidium biology. Cryptosporidium spp. are important diarrhea-associated pathogens in humans and livestock. Among the known species, Cryptosporidium xiaoi , which causes cryptosporidiosis in sheep and goats, was previously recognized as a genotype of the bovine-specific Cryptosporidium bovis based on their high sequence identity in the ssrRNA gene. However, the lack of genomic data has limited characterization of the genetic differences between the two closely related species. In this study, we sequenced the genomes of two C. xiaoi isolates and performed comparative genomic analysis to identify the sequence uniqueness of this ovine-adapted species compared with other Cryptosporidium spp. Our results showed that C. xiaoi is genetically related to C. bovis as shown by their 95.8% genomic identity and similar gene content. Consistent with this, both C. xiaoi and C. bovis appear to have fewer genes encoding mitochondrial metabolic enzymes and invasion-related protein families. However, they appear to possess several species-specific genes. Further analysis indicates that the sequence differences between these two Cryptosporidium spp. are mainly in 24 highly polymorphic genes, half of which are located in the subtelomeric regions. Some of these subtelomeric genes encode secretory proteins that have undergone positive selection. In addition, the genomes of two C. xiaoi isolates, identified as subtypes XXIIIf and XXIIIh, share 99.9% nucleotide sequence identity, with six highly divergent genes encoding putative secretory proteins. Therefore, these species-specific genes and sequence polymorphism in subtelomeric genes probably contribute to the different host preference of C. xiaoi and C. bovis. [ABSTRACT FROM AUTHOR]

Background: Caused by duplications of the gene encoding peripheral myelin protein 22 (PMP22), Charcot–Marie–Tooth disease type 1A (CMT1A) is the most common hereditary neuropathy. Despite this shared genetic origin, there is considerable variability in clinical severity. It is hypothesized that genetic modifiers contribute to this heterogeneity, the identification of which may reveal novel therapeutic targets. In this study, we present a comprehensive analysis of clinical examination results from 1564 CMT1A patients sourced from a prospective natural history study conducted by the RDCRN‐INC (Inherited Neuropathy Consortium). Our primary objective is to delineate extreme phenotype profiles (mild and severe) within this patient cohort, thereby enhancing our ability to detect genetic modifiers with large effects. Methods: We have conducted large‐scale statistical analyses of the RDCRN‐INC database to characterize CMT1A severity across multiple metrics. Results: We defined patients below the 10th (mild) and above the 90th (severe) percentiles of age‐normalized disease severity based on the CMT Examination Score V2 and foot dorsiflexion strength (MRC scale). Based on extreme phenotype categories, we defined a statistically justified recruitment strategy, which we propose to use in future modifier studies. Interpretation: Leveraging whole genome sequencing with base pair resolution, a future genetic modifier evaluation will include single nucleotide association, gene burden tests, and structural variant analysis. The present work not only provides insight into the severity and course of CMT1A, but also elucidates the statistical foundation and practical considerations for a cost‐efficient and straightforward patient enrollment strategy that we intend to conduct on additional patients recruited globally. [ABSTRACT FROM AUTHOR]

Aims To investigate alternative resistance mechanisms among seven ceftazidime–avibactam (CZA)-resistant carbapenem-resistant Klebsiella pneumoniae (CRKP) strains lacking common antimicrobial resistance genes (ARGs) using whole genome sequencing. Methods and Results ARG and virulence factors (VFs) were screened using the ARG database CARD and the VF database, respectively, and identified using genomic annotation data with BLAST+. Six strains were ST11 sequence types (STs), and one was ST2123. ST11 strains harbored more ARGs than the ST2123 strains. All seven strains carried multiple ARGs with efflux-mediated antibiotic resistance, including oqxA, oqxB, tet (A), qacEdltal, CRP, H-NS, Kpn-E, F, G, H, acrA, LptD, acrB, acrD, cpxA, mdtB, and mdtC. These efflux-mediated ARGs were identified in most strains and even all strains. Whole genome sequencing revealed that the ST11 strain carried multiple potential prophages, genomic islands, and integrative and conjugative elements, while the ST2123 strain carried an independent potential prophages and a genomic island. Conclusions Whole genome sequencing analysis revealed that these seven CZA-resistant CRKP strains lacking common ARGs exhibited efflux-mediated antibiotic resistance-associated ARGs. The main mechanism by which CRKP resists CZA is antibiotic inactivation. Except for tet (A), no ARGs and validation experiments related to efflux were found. This study's results provide a new possibility for the resistance mechanism of CRKP to CZA, and we will verify this conclusion through experiments in the future. [ABSTRACT FROM AUTHOR]

Spontaneous tumour formation in higher plants can occur in the absence of pathogen invasion, depending on the plant genotype. Spontaneous tumour formation on the taproots is consistently observed in certain inbred lines of radish (Raphanus sativus var. radicula Pers.). In this paper, using Oxford Nanopore and Illumina technologies, we have sequenced the genomes of two closely related radish inbred lines that differ in their ability to spontaneously form tumours. We identified a large number of single nucleotide variants (amino acid substitutions, insertions or deletions, SNVs) that are likely to be associated with the spontaneous tumour formation. Among the genes involved in the trait, we have identified those that regulate the cell cycle, meristem activity, gene expression, and metabolism and signalling of phytohormones. After identifying the SNVs, we performed Sanger sequencing of amplicons corresponding to SNV-containing regions to validate our results. We then checked for the presence of SNVs in other tumour lines of the radish genetic collection and found the ERF118 gene, which had the SNVs in the majority of tumour lines. Furthermore, we performed the identification of the CLAVATA3/ESR (CLE) and WUSCHEL (WOX) genes and, as a result, identified two unique radish CLE genes which probably encode proteins with multiple CLE domains. The results obtained provide a basis for investigating the mechanisms of plant tumour formation and also for future genetic and genomic studies of radish. [ABSTRACT FROM AUTHOR]

Promoter trapping is a powerful tool for discovering promoters and uses promoter trapping vectors. However, the traditional trapping vector allows expression even if it does not integrate into the host cell genome, and even if it does integrate into the genome, it is more likely to integrate in a region other than the promoter region. In this study, to overcome the shortcomings of traditional trapping vectors, we used the bicistronic 2A system to link GFP and the neomycin resistance gene. Because this vector does not contain a promoter, simultaneous production of GFP and neomycin resistance protein requires integration into the promoter region. In fact, GFP expression was observed in more than 90% of the cell clones that survived in the medium containing antibiotics, confirming that the 2A system operates. The vector insertion location was confirmed through whole genome sequence analysis, and a 1-kb promoter candidate region was selected through promoter motif analysis. In fact, a 1-kb region inserted into a promoterless luciferase expression vector showed strong promoter activity, demonstrating its utility as a tool to find promoters. In summary, we constructed a novel promoter trapping vector using the 2A system and used it to discover the promoter with strong activity. This vector will increase the efficiency of promoter trapping, providing an opportunity to easily discover new promoters in mammalian cells. [ABSTRACT FROM AUTHOR]

The current study evaluates antibiotic susceptibility and Extended Spectrum β-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 β-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 β-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. [ABSTRACT FROM AUTHOR]

We analyzed antimicrobial resistance and virulence traits in multidrug-resistant (MDR) E. coli isolates obtained from imported shrimp using whole-genome sequences (WGSs). Antibiotic resistance profiles were determined phenotypically. WGSs identified key characteristics, including their multilocus sequence type (MLST), serotype, virulence factors, antibiotic resistance genes, and mobile elements. Most of the isolates exhibited resistance to gentamicin, streptomycin, ampicillin, chloramphenicol, nalidixic acid, ciprofloxacin, tetracycline, and trimethoprim/sulfamethoxazole. Multilocus sequence type (MLST), serotype, average nucleotide identity (ANI), and pangenome analysis showed high genomic similarity among isolates, except for EC15 and ECV01. The EC119 plasmid contained a variety of efflux pump genes, including those encoding the acid resistance transcriptional activators (gadE, gadW, and gadX), resistance-nodulation-division-type efflux pumps (mdtE and mdtF), and a metabolite, H1 symporter (MHS) family major facilitator superfamily transporter (MNZ41_23075). Virulence genes displayed diversity, particularly EC15, whose plasmids carried genes for adherence (faeA and faeC-I), invasion (ipaH and virB), and capsule (caf1A and caf1M). This comprehensive analysis illuminates antimicrobial resistance, virulence, and plasmid dynamics in E. coli from imported shrimp and has profound implications for public health, emphasizing the need for continued surveillance and research into the evolution of these important bacterial pathogens. [ABSTRACT FROM AUTHOR]

Seventeen Gram-negative, facultatively anaerobic bacterial strains were isolated from bleeding cankers of various broadleaf hosts and oak rhizosphere soil in Great Britain. The strains were tentatively identified as belonging to the genus Raoultella based on 16S rRNA gene sequencing. Multilocus sequence analysis (MLSA), based on four protein-encoding genes (fusA, leuS, pyrG, and rpoB), separated the strains into three clusters within the Raoultella genus clade. The majority of strains clustered with the type strain of Raoultella terrigena, with the remaining strains divided into two clusters with no known type strain. Whole genome sequencing comparisons confirmed these two clusters of strains as belonging to two novel Raoultella species which can be differentiated phenotypically from their current closest phylogenetic relatives. Therefore, two novel species are proposed: Raoultella scottia sp. nov. (type strain = BAC 10a-01-01T = LMG 33072T = CCUG 77096T) and Raoultella lignicola sp. nov. (type strain = TW_WC1a.1T = LMG 33073T = CCUG 77094T). [ABSTRACT FROM AUTHOR]

Background: Shallots are infected by various viruses like Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and Shallot virus X (ShVX). In India, they have been found to be persistently infected by ShVX. ShVX also infects onion and garlic in combination with other carlaviruses and potyviruses. ShVX is a member of genus Allexivirus of family Alphaflexiviridae. ShVX has a monopartite genome, which is represented by positive sense single-stranded RNA. Globally, only six complete and 3 nearly complete genome sequences of ShV X are reported to date. This number is insufficient to measure a taxon's true molecular diversity. Moreover, the complete genome sequence of ShVX from Asia has not been reported as yet. Therefore, this study was undertaken to generate a complete genome sequence of ShVX from India. Results: Shallot virus X (ShVX) is one of the significant threats to Allium crop production. In this study, we report the first complete genome sequence of the ShVX from India through Next-generation sequencing (NGS). The complete genome of the ShVX (Accession No. OK104171), from this study comprised 8911 nucleotides. In-silico analysis of the sequence revealed variability between this isolate and isolates from other countries. The dissimilarities are spread all over the genome specifically some non-coding intergenic regions. Statistical analysis of individual genes for site-specific selection indicates a positive selection in NABP region. The presence of a recombination event was detected in coat protein region. The sequence similarity percentage and phylogenetic analysis indicate ShVX Indian isolate is a distinctly different isolate. Recombination and site-specific selection may have a function in the evolution of this isolate. This is the first detailed study of the ShVX complete genome sequence from Southeast Asia. Conclusion: This study presents the first report of the entire genome sequence of an Indian isolate of ShVX along with an in-depth exploration of its evolutionary traits. The findings highlight the Indian variant as a naturally occurring recombinant, emphasizing the substantial role of recombination in the evolution of this viral species. This insight into the molecular diversity of strains within a specific geographical region holds immense significance for comprehending and forecasting potential epidemics. Consequently, the insights garnered from this research hold practical value for shaping ShVX management strategies and providing a foundation for forthcoming studies delving into its evolutionary trajectory. [ABSTRACT FROM AUTHOR]

Introduction: Serratia marcescens is an opportunistic pathogen found ubiquitously in the environment and associated with a wide range of nosocomial infections. This multidrug-resistant bacterium has been a cause of concern for hospitals and healthcare facilities due to its ability to spread rapidly and cause outbreaks. Next generation sequencing genotyping of bacterial isolates has proven to be a valuable tool for tracking the spread and transmission of nosocomial infections. This has allowed for the identification of outbreaks and transmission chains, as well as determining whether cases are due to endogenous or exogenous sources. Evidence of nosocomial transmission has been gathered through genotyping methods. The aim of this study was to investigate the genetic diversity of carbapenemase-producing S. marcescens in an outbreak at a public hospital in Cuiaba, MT, Brazil. Methodology: Ten isolates of S. marcenses were sequenced and antibiotic resistance profiles analyzed over 12 days. Results: The isolates were clonal and multidrug resistant. Gentamycin and tigecycline had sensitivity in 90% and 80% isolates, respectively. Genomic analysis identified several genes that encode β-lactamases, aminoglycoside-modifying enzymes, efflux pumps, and other virulence factors. Conclusions: Systematic surveillance is crucial in monitoring the evolution of S. marcescens genotypes, as it can lead to early detection and prevention of outbreaks. [ABSTRACT FROM AUTHOR]

Bacillus paralicheniformis, a species known to produce the antimicrobial bacitracin, could be misidentified as Bacillus licheniformis, depending on the identification method used. For this reason, the European Commission requested EFSA to review the taxonomic identification of formerly assessed B. licheniformis production strains. Following this request, EFSA retrieved the raw data from 27 technical dossiers submitted and found that the taxonomic identification was established by 16S rRNA gene analyses for 15 strains and by whole genome sequence analysis for 12 strains. As a conclusion, only these 12 strains could be unambiguously identified as B. licheniformis. [ABSTRACT FROM AUTHOR]

Objectives: The identification of structural variants and single‐nucleotide variants is essential in finding molecular etiologies of monogenic genetic disorders. Whole‐genome sequencing (WGS) is becoming more widespread in genetic disease diagnosis. However, data on its clinical utility remain limited in prenatal practice. We aimed to expand our understanding of implementing WGS in the genetic diagnosis of fetal structural anomalies. Methods: We employed trio WGS with a minimum coverage of 40× on the MGI DNBSEQ‐T7 platform in a cohort of 17 fetuses presenting with aberrations detected by ultrasound, but uninformative findings of standard chromosomal microarray analysis (CMA) and exome sequencing (ES). Results: Causative genetic variants were identified in two families, with an increased diagnostic yield of 11.8% (2/17). Both were exon‐level copy‐number variants of small size (3.03 kb and 5.16 kb) and beyond the detection thresholds of CMA and ES. Moreover, to the best of our knowledge, we have described the first prenatal instance of the association of FGF8 with holoprosencephaly and facial deformities. Conclusions: Our analysis demonstrates the clinical value of WGS in the diagnosis of the underlying etiology of fetuses with structural abnormalities, when routine genetic tests have failed to provide a diagnosis. Additionally, the novel variants and new fetal manifestations have expanded the mutational and phenotypic spectrums of BBS9 and FGF8. © 2023 International Society of Ultrasound in Obstetrics and Gynecology. [ABSTRACT FROM AUTHOR]

Importance: The emergence and rapid increase in the incidence of multidrug-resistant (MDR) bacteria in pig farms has become a serious concern and reduced the choice of effective antibiotics. Objective: This study analyzed the phylogenetics and diversity of antibiotic resistance genes (ARGs) and molecularly identified the source of ARGs in antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia. Methods: Forty-four antibiotic-resistant E. coli isolates from fecal samples from 44 pig farms in Banten Province, Indonesia, were used as samples. The samples were categorized into 14 clusters. Sequencing was performed using the Oxford Nanopore Technologies MinION platform, with barcoding before sequencing with Nanopore Rapid sequencing gDNAbarcoding (SQK-RBK110.96) according to manufacturing procedures. ARG detection was conducted using ResFinder, and the plasmid replicon was determined using PlasmidFinder. Results: Three phylogenetic leaves of E. coli were identified in the pig farming cluster in Banten Province. The E. coli isolates exhibited potential resistance to nine classes of antibiotics. Fiftyone ARGs were identified across all isolates, with each cluster carrying a minimum of 10 ARGs. The ant(3")-Ia and qnrS1 genes were present in all isolates. ARGs in the E. coli pig farming cluster originated mainly from plasmids, accounting for an average of 89.4%. Conclusions and Relevance: The elevated potential for MDR events, coupled with the dominance of ARGs originating from plasmids, increases the risk of ARG spread among bacterial populations in animals, humans, and the environment. [ABSTRACT FROM AUTHOR]

The rice receptor kinase XA21 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight disease. To investigate the relationship between the expression level of XA21 and resulting resistance, we generated independent HA-XA21 transgenic rice lines accumulating the XA21 immune receptor fused with an HA epitope tag. Whole-genome sequence analysis identified the T-DNA insertion sites in sixteen independent T0 events. Through quantification of the HA-XA21 protein and assessment of the resistance to Xoo strain PXO99 in six independent transgenic lines, we observed that XA21-mediated resistance is dose dependent. In contrast, based on the four agronomic traits quantified in these experiments, yield is unlikely to be affected by the expression level of HA-XA21. These findings extend our knowledge of XA21-mediated defense and contribute to the growing number of well-defined genomic landing pads in the rice genome that can be targeted for gene insertion without compromising yield. [ABSTRACT FROM AUTHOR]

Asplenium antiquum Makino 1929 is one of the Endangered endemic species on the Korean Peninsula. The complete chloroplast of A. antiquum is 150,690 bp in length with typical quadripartite structure comprised of large single-copy region of (83,166 bp), a small single copy region (21,932 bp), and two inverted repeat regions, each 22,796 bp in length. 114 genes were detected in the chloroplast genome of A. antiquum, comprising 84 protein-encoding genes, 26 tRNA genes, and 4 rRNA genes. The phylogenetic analysis revealed a monophyletic relationship, placing A. antiquum as a sister to voth A. Prolongatum and A. nidus, forming a subclade of Asplenium species within the Aspleniaceae family. The genomic data obtained from this study will serve as valuable information for the species' genetic classification of Asplenium. [ABSTRACT FROM AUTHOR]

Tylopilus brunneirubens is a common species in southern China. It is known for brown to dark brown pileus, white context turning reddish brown or rust brown when touched and distinct reticulation on the upper stem. However, little is known about its mitochondrial genome and its relationship with other boletes. Our analysis revealed that the mitochondrial genome of this species is a circular DNA molecule that spans 32,389 bp. It contains 15 core protein-coding genes, 24 transfer RNA genes, and two ribosomal RNA genes. The base composition of the mitochondrial genome is as follows: A (37.20%), C (11.32%), G (12.48%), and T (39.00%), with a GC content of 23.80%. Furthermore, a phylogenetic tree based on 24 mitochondrial genomes provided valuable insights into the phylogenetic relationships of Tylopilus brunneirubens with other boletes for the first time. [ABSTRACT FROM AUTHOR]

Chlorophytum laxum of Asparagaceae is a valuable ornamental plant native to the tropical regions of Asia, Africa, and Australia. The plant also has medicinal properties and is used as source for folk medicine. Despite being commercially important, genetic studies of C. laxum are still limited. To expand the genomic information of this plant species, we sequenced, assembled, and characterized its complete chloroplast genome. The chloroplast genome was 153,678 bp in length, with a large single-copy region (83,225 bp) and a small single-copy region (18,031 bp) separated by a pair of inverted repeat regions (26,211 bp each). A total of 127 genes were predicted, including 81 protein-coding, 38 tRNA, and eight rRNA genes. The overall GC content was 37.3%. Based on current sampling size, phylogenetic analysis using the maximum likelihood based on the complete chloroplast genome sequence revealed that the relationship in Chlorophytum is well resolved; C. laxum was closely related to C. rhizopendulum. [ABSTRACT FROM AUTHOR]

The study used whole-genome sequencing (WGS) and bioinformatics analysis for the genomic characterization of 60 isolates of Listeria monocytogenes obtained from the beef production chain (cattle farms, abattoirs, and retail outlets) in Gauteng province, South Africa. The sequence types (STs), clonal complexes (CCs), and the lineages of the isolates were determined using in silico multilocus sequence typing (MLST). We used BLAST-based analyses to identify virulence and antimicrobial genes, plasmids, proviruses/prophages, and the CRISPR-Cas system. The study investigated any association of the detected genes to the origin in the beef production chain of the L. monocytogenes isolates. Overall, in 60 isolates of Listeria monocytogenes, there were seven STs, six CCs, forty-four putative virulence factors, two resistance genes, one plasmid with AMR genes, and three with conjugative genes, one CRISPR gene, and all 60 isolates were positive for proviruses/prophages. Among the seven STs detected, ST204 (46.7%) and ST2 (21.7%) were the most prominent, with ST frequency varying significantly (p < 0.001). The predominant CC detected were CC2 (21.7%) and CC204 (46.7%) in lineages I and II, respectively. Of the 44 virulence factors detected, 26 (across Listeria Pathogenicity Islands, LIPIs) were present in all the isolates. The difference in the detection frequency varied significantly (p < 0.001). The two AMR genes (fosX and vga(G)) detected were present in all 60 (100%) isolates of L. monocytogenes. The only plasmid, NF033156, was present in three (5%) isolates. A CRISPR-Cas system was detected in six (10%), and all the isolates carried proviruses/prophages. The source and sample type significantly affected the frequencies of STs and virulence factors in the isolates of L. monocytogenes. The presence of fosX and vga(G) genes in all L. monocytogenes isolates obtained from the three industries of the beef production chain can potentially cause therapeutic implications. Our study, which characterized L. monocytogenes recovered from the three levels in the beef production chain, is the first time genomics was performed on this type of data set in the country, and this provides insights into the health implications of Listeria. [ABSTRACT FROM AUTHOR]

The present study involves the precise identification and safety evaluation of Enterococcus casseliflavus KB1733, previously identified using 16S rRNA analysis, through whole-genome sequencing, phenotypic analysis, and preclinical toxicity studies. Analyses based on the genome sequencing data confirm the identity of KB1733 as E. casseliflavus and show that the genes related to vancomycin resistance are only present on the chromosome, while no virulence factor genes are present on the chromosome or plasmid. Phenotypic analyses of antibiotic resistance and hemolytic activity also indicated no safety concerns. A bacterial reverse mutation test showed there was no increase in revertant colonies of heat-killed KB1733. An acute toxicity test employing heat-killed KB1733 at a dose of 2000 mg/kg body weight in rats resulted in no deaths and no weight gain or other abnormalities in the general condition of the animals, with renal depression foci and renal cysts only occurring at the same frequency as in the control. Taking the background data into consideration, the effects on the kidneys observed in the current study were not caused by KB1733. Our findings suggest that KB1733 is non-pathogenic to humans/animals, although further studies involving repeated oral toxicity tests and/or clinical tests are required. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Salmonella enterica (S. enterica) pose a significant threat to public health, causing gastroenteritis and invasive infections. We report the first emergence of a carbapenem-resistant S. enterica serovar London strain, A132, carrying the blaNDM-5 gene in China. Whole-genome sequencing and bioinformatics analysis assigned A132 to be ST155, a multidrug-resistant clone frequently reported in China. The strain A132 exhibited resistance to multiple antibiotics, with 20 acquired antibiotic resistance genes (ARGs) identified, predominantly located on the IncFIB plasmid (pA132-1-NDM). Notably, the blaNDM-5 gene was located within an IS 26 flanked-class 1 integron-ISCR1 complex, comprising two genetic cassettes. One cassette is the class 1 integron, which may facilitate the transmission of the entire complex, while the other is the blaNDM-5-containing ISCR1-IS 26-flanked cassette, carrying multiple other ARGs. Genbank database search based on the blaNDM-5-carrying cassette identified a similar genetic context found in transmissible IncFIA plasmids from Escherichia coli (p91) and Enterobacter hormaechei (p388) with a shared host range, suggesting the potential for cross-species transmission of blaNDM-5. To our knowledge, this is the first reported case of Salmonella serovar London ST155 harboring blaNDM-5 gene. Phylogenetic analysis indicated a close relationship between A132 and eight S. London ST155 strains isolated from the same province. However, A132 differed by carrying the blaNDM-5 gene and four unique ARGs. Given the high transmissibility of the F-type plasmid harboring blaNDM-5 and 18 other ARGs, it is imperative to implement vigilant surveillance and adopt appropriate infection control measures to mitigate the threat to public health. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) causes liver diseases (chronic hepatitis, cirrhosis, and hepatocellular carcinoma) and is a leading health problem worldwide. Sequencing of the whole HBV genome provides insight into the virus genotype, subgenotype, serotype, genetic variation, and viral drug resistance. To date, no study has been conducted on the whole genome sequence of HBV obtained from Iraqi patients. Therefore, this is the first study to sequence clinical samples from these patients. Viral genomic DNA was isolated and amplified using five primer sets to amplify five overlapping regions covering the entire HBV genome. The amplicons were sequenced, aligned to a reference sequence, annotated, and submitted to the National Center for Biotechnology Information GenBank database. Sequence analysis showed that the genome size of the tested viral samples was 3,182 bp and belonged to genotype D, subgenotype D1, and serotype ayw2. Missense mutations were found in the four regions (X, PreS1-S, PreC-C, and P) of the tested samples, leading to amino acid substitutions, which were 8.4%, 5.1%, 4.7%, and 4.6%, respectively. These mutations may cause severe liver diseases. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, a high-quality chromosomal-level genome of male Decapterus maruadsi was assembled based on Illumina, PacBio, and Hi-C technology. Notably, 23 chromosome-level genome sequences with lengths ranging between 21.74 and 44.53 Mb were assembled. A total of 22,716 protein-coding genes with an average transcript and a CDs length of 12,823.03 bp and 1676.66 bp, respectively, were successfully annotated. Based on positive selection analysis, some genes associated with the growth and development of bone, muscle, cardioid, and ovary were screened. These genes were likely involved in the evolution of typical biological features in D. maruadsi, such as fast growth rate, small body size, and strong fecundity. The newly established reference genome provides a fundamental genome resource for further genetic conservation, genomic-assisted breeding, and exploration of the molecular mechanism underlying adaptive evolution. Decapterus maruadsi is a typical representative of small pelagic fish characterized by fast growth rate, small body size, and high fecundity. It is a high-quality marine commercial fish with high nutritional value. However, the underlying genetics and genomics research focused on D. maruadsi is not comprehensive. Herein, a high-quality chromosome-level genome of a male D. maruadsi was assembled. The assembled genome length was 716.13 Mb with contig N50 of 19.70 Mb. Notably, we successfully anchored 95.73% contig sequences into 23 chromosomes with a total length of 685.54 Mb and a scaffold N50 of 30.77 Mb. A total of 22,716 protein-coding genes, 274.90 Mb repeat sequences, and 10,060 ncRNAs were predicted, among which 22,037 (97%) genes were successfully functionally annotated. The comparative genome analysis identified 459 unique, 73 expanded, and 52 contracted gene families. Moreover, 2804 genes were identified as candidates for positive selection, of which some that were related to the growth and development of bone, muscle, cardioid, and ovaries, such as some members of the TGF-β superfamily, were likely involved in the evolution of typical biological features in D. maruadsi. The study provides an accurate and complete chromosome-level reference genome for further genetic conservation, genomic-assisted breeding, and adaptive evolution research for D. maruadsi. [ABSTRACT FROM AUTHOR]

P1 is a model, temperate bacteriophage of the 94 kb genome. It can lysogenize representatives of the Enterobacterales order. In lysogens, it is maintained as a plasmid. We tested P1 interactions with the biocontrol P. agglomerans L15 strain to explore the utility of P1 in P. agglomerans genome engineering. A P1 derivative carrying the Tn9 (cmR) transposon could transfer a plasmid from Escherichia coli to the L15 cells. The L15 cells infected with this derivative formed chloramphenicol-resistant colonies. They could grow in a liquid medium with chloramphenicol after adaptation and did not contain prophage P1 but the chromosomally inserted cmR marker of P1 Tn9 (cat). The insertions were accompanied by various rearrangements upstream of the Tn9 cat gene promoter and the loss of IS1 (IS1L) from the corresponding region. Sequence analysis of the L15 strain genome revealed a chromosome and three plasmids of 0.58, 0.18, and 0.07Mb. The largest and the smallest plasmid appeared to encode partition and replication incompatibility determinants similar to those of prophage P1, respectively. In the L15 derivatives cured of the largest plasmid, P1 with Tn9 could not replace the smallest plasmid even if selected. However, it could replace the smallest and the largest plasmid of L15 if its Tn9 IS1L sequence driving the Tn9 mobility was inactivated or if it was enriched with an immobile kanamycin resistance marker. Moreover, it could develop lytically in the L15 derivatives cured of both these plasmids. Clearly, under conditions of selection for P1, the mobility of the P1 selective marker determines whether or not the incoming P1 can outcompete the incompatible L15 resident plasmids. Our results demonstrate that P. agglomerans can serve as a host for bacteriophage P1 and can be engineered with the help of this phage. They also provide an example of how antibiotics can modify the outcome of horizontal gene transfer in natural environments. Numerous plasmids of Pantoea strains appear to contain determinants of replication or partition incompatibility with P1. Therefore, P1 with an immobile selective marker may be a tool of choice in curing these strains from the respective plasmids to facilitate their functional analysis. [ABSTRACT FROM AUTHOR]

Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples ("Neethling-like" clade 1.1 and "Kenya-like" subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies. [ABSTRACT FROM AUTHOR]

Diagnostic laboratories in Aotearoa, New Zealand (NZ) refer cultures from faecal samples positive for Shiga toxin genes to the national Enteric Reference Laboratory for isolation of Shiga toxin-producing Escherichia coli (STEC) for epidemiological typing. As there was variation in the culture media being referred, a panel of 75 clinical isolates of STEC, representing 28 different serotypes, was used to assess six commercially available media and provide guidance to clinical laboratories. Recommendations were subsequently tested for a 3-month period, where STEC isolations and confirmations were assessed by whole genome sequencing analysis against the culture media referred. CHROMagar™ STEC (CH-STEC; CHROMagar Microbiology, Paris, France) or CH-STEC plus cefixime-tellurite sorbitol MacConkey agar was confirmed inferior to CH-STEC plus blood agar with vancomycin, cefsulodin, and cefixime (BVCC). The former resulted in fewer STEC types (n  = 18) being confirmed compared to those from a combination of CH-STEC and BVCC (n  = 42). A significant (P  < .05) association with an STEC's ability to grow on CH-STEC and the presence of the ter gene cluster, and eae was observed. Culturing screen positive STEC samples onto both CH-STEC and BVCC ensures a consistently higher recovery of STEC from all clinical samples in NZ than CH-STEC alone. [ABSTRACT FROM AUTHOR]

Infertility is a global health challenge that affects an estimated 72.4 million people worldwide. Between 30 and 50% of these cases involve male factors, showcasing the complex nature of male infertility, which can be attributed to both environmental and genetic determinants. Asthenozoospermia, a condition characterized by reduced sperm motility, stands out as a significant contributor to male infertility. This study explores the involvement of the mitochondrial oxidative phosphorylation (OXPHOS) system, crucial for ATP production and sperm motility, in asthenozoospermia. Through whole-genome sequencing and in silico analysis, our aim was to identify and characterize OXPHOS gene variants specific to individuals with asthenozoospermia. Our analysis identified 680,099 unique variants, with 309 located within OXPHOS genes. Nine of these variants were prioritized due to their significant implications, such as potential associations with diseases, effects on gene expression, protein function, etc. Interestingly, none of these variants had been previously associated with male infertility, opening up new avenues for research. Thus, through our comprehensive approach, we provide valuable insights into the genetic factors that influence sperm motility, laying the foundation for future research in the field of male infertility. [ABSTRACT FROM AUTHOR]

Knowledge of genetic diversity and structure is essential for developing conservation strategies for endangered species. Blue whales were hunted to near extinction in the mid-twentieth century. Not-withstanding almost 380,000 animals killed globally, much remains unknown about their population structure and migration patterns. Herein, we use whole genome sequencing to elucidate the poorly understood population genetics of North Atlantic (NA) blue whales. We generated a de novo genome assembly for a NA blue whale to analyze 19 other whole genomic sequences and 31 complete mitochondrial genomes. Present-day and historical samples (earliest from 1876) from the Atlantic and Antarctic Oceans were included to understand the impact of whaling on the genetic diversity of this species. We found low but statistically significant population structuring and high genetic diversity. Demographic modeling using fastsimcoal2 rejected an absence of gene flow between eastern and western NA blue whales and suggested an asymmetric west to east gene flow. Introgression estimated using D-statistics between blue and fin whales (Balaenoptera physalus), was observed in all present-day samples. This gene flow was found to be unidirectional from fin whales to blue whales and accounts for ~ 3.5% of the NA blue whale's genome. Our sequencing and population structure analyses provide a genomic baseline to inform ongoing conservation strategies for this iconic species. [ABSTRACT FROM AUTHOR]