SNP-SVant: A Computational Workflow to Predict and Annotate Genomic Variants in Organisms Lacking Benchmarked Variants.

Whole-genome sequencing is widely used to investigate population genomic variation in organisms of interest. Assorted tools have been independently developed to call variants from short-read sequencing data aligned to a reference genome, including single nucleotide polymorphisms (SNPs) and structural variations (SVs). We developed SNP-SVant, an integrated, flexible, and computationally efficient bioinformatic workflow that predicts high-confidence SNPs and SVs in organisms without benchmarked variants, which are traditionally used for distinguishing sequencing errors from real variants. In the absence of these benchmarked datasets, we leverage multiple rounds of statistical recalibration to increase the precision of variant prediction. The SNP-SVant workflow is flexible, with user options to tradeoff accuracy for sensitivity. The workflow predicts SNPs and small insertions and deletions using the Genome Analysis ToolKit (GATK) and predicts SVs using the Genome Rearrangement IDentification Software Suite (GRIDSS), and it culminates in variant annotation using custom scripts. A key utility of SNP-SVant is its scalability. Variant calling is a computationally expensive procedure, and thus, SNP-SVant uses a workflow management system with intermediary checkpoint steps to ensure efficient use of resources by minimizing redundant computations and omitting steps where dependent files are available. SNP-SVant also provides metrics to assess the quality of called variants and converts between VCF and aligned FASTA format outputs to ensure compatibility with downstream tools to calculate selection statistics, which are commonplace in population genomics studies. By accounting for both small and large structural variants, users of this workflow can obtain a wide-ranging view of genomic alterations in an organism of interest. Overall, this workflow advances our capabilities in assessing the functional consequences of different types of genomic alterations, ultimately improving our ability to associate genotypes with phenotypes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Predicting single nucleotide polymorphisms and structural variations Support Protocol 1: Downloading publicly available sequencing data Support Protocol 2: Visualizing variant loci using Integrated Genome Viewer Support Protocol 3: Converting between VCF and aligned FASTA formats.

Whole-genome analysis of Escherichia coli isolated from wild Amur tiger (Panthera tigris altaica) and North China leopard (Panthera pardus japonensis).

Background: Escherichia coli is an important intestinal flora, of which pathogenic E. coli is capable of causing many enteric and extra-intestinal diseases. Antibiotics are essential for the treatment of bacterial infections caused by pathogenic E. coli; however, with the widespread use of antibiotics, drug resistance in E. coli has become particularly serious, posing a global threat to human, animal, and environmental health. While the drug resistance and pathogenicity of E. coli carried by tigers and leopards in captivity have been studied intensively in recent years, there is an extreme lack of information on E. coli in these top predators in the wild environment. Methods: Whole genome sequencing data of 32 E. coli strains collected from the feces of wild Amur tiger (Panthera tigris altaica, n = 24) and North China leopard (Panthera pardus japonensis, n = 8) were analyzed in this article. The multi-locus sequence types, serotypes, virulence and resistance genotypes, plasmid replicon types, and core genomic SNPs phylogeny of these isolates were studied. Additionally, antimicrobial susceptibility testing (AST) was performed on these E. coli isolates. Results: Among the E. coli isolates studied, 18 different sequence types were identified, with ST939 (21.9%), ST10 (15.6%), and ST3246 (9.4%) being the most prevalent. A total of 111 virulence genes were detected, averaging about 54 virulence genes per sample. They contribute to invasion, adherence, immune evasion, efflux pump, toxin, motility, stress adaption, and other virulence-related functions of E. coli. Sixty-eight AMR genes and point mutations were identified. Among the detected resistance genes, those belonging to the efflux pump family were the most abundant. Thirty-two E. coli isolates showed the highest rate of resistance to tetracycline (14/32; 43.8%), followed by imipenem (4/32; 12.5%), ciprofloxacin (3/32; 9.4%), doxycycline (2/32; 6.3%), and norfloxacin (1/32; 3.1%). Conclusions: Our results suggest that E. coli isolates carried by wild Amur tigers and North China leopards have potential pathogenicity and drug resistance.

SARS-CoV-2 variant introduction following spring break travel and transmission mitigation strategies.

BACKGROUND: University spring break carries a two-pronged SARS-CoV-2 variant transmission risk. Circulating variants from universities can spread to spring break destinations, and variants from spring break destinations can spread to universities and surrounding communities. Therefore, it is critical to implement SARS-CoV-2 variant surveillance and testing strategies to limit community spread before and after spring break to mitigate virus transmission and facilitate universities safely returning to in-person teaching. METHODS: We examined the SARS-CoV-2 positivity rate and changes in variant lineages before and after the university spring break for two consecutive years. 155 samples were sequenced across four time periods: pre- and post-spring break 2021 and pre- and post-spring break 2022; following whole genome sequencing, samples were assigned clades. The clades were then paired with positivity and testing data from over 50,000 samples. RESULTS: In 2021, the number of variants in the observed population increased from four to nine over spring break, with variants of concern being responsible for most of the cases; Alpha percent composition increased from 22.2% to 56.4%. In 2022, the number of clades in the population increased only from two to three, all of which were Omicron or a sub-lineage of Omicron. However, phylogenetic analysis showed the emergence of distantly related sub-lineages. 2022 saw a greater increase in positivity than 2021, which coincided with a milder mitigation strategy. Analysis of social media data provided insight into student travel destinations and how those travel events may have impacted spread. CONCLUSIONS: We show the role that repetitive testing can play in transmission mitigation, reducing community spread, and maintaining in-person education. We identified that distantly related lineages were brought to the area after spring break travel regardless of the presence of a dominant variant of concern.

Copy number variation and elevated genetic diversity at immune trait loci in Atlantic and Pacific herring.

BACKGROUND: Genome-wide comparisons of populations are widely used to explore the patterns of nucleotide diversity and sequence divergence to provide knowledge on how natural selection and genetic drift affect the genome. In this study we have compared whole-genome sequencing data from Atlantic and Pacific herring, two sister species that diverged about 2 million years ago, to explore the pattern of genetic differentiation between the two species. RESULTS: The genome comparison of the two species revealed high genome-wide differentiation but with islands of remarkably low genetic differentiation, as measured by an FST analysis. However, the low FST observed in these islands is not caused by low interspecies sequence divergence (dxy) but rather by exceptionally high estimated intraspecies nucleotide diversity (π). These regions of low differentiation and elevated nucleotide diversity, termed high-diversity regions in this study, are not enriched for repeats but are highly enriched for immune-related genes. This enrichment includes genes from both the adaptive immune system, such as immunoglobulin, T-cell receptor and major histocompatibility complex genes, as well as a substantial number of genes with a role in the innate immune system, e.g. novel immune-type receptor, tripartite motif and tumor necrosis factor receptor genes. Analysis of long-read based assemblies from two Atlantic herring individuals revealed extensive copy number variation in these genomic regions, indicating that the elevated intraspecies nucleotide diversities were partially due to the cross-mapping of short reads. CONCLUSIONS: This study demonstrates that copy number variation is a characteristic feature of immune trait loci in herring. Another important implication is that these loci are blind spots in classical genome-wide screens for genetic differentiation using short-read data, not only in herring, likely also in other species harboring qualitatively similar variation at immune trait loci. These loci stood out in this study because of the relatively high genome-wide baseline for FST values between Atlantic and Pacific herring.

Characterization of linezolid- and methicillin-resistant coagulase-negative staphylococci in a tertiary hospital in China.

BACKGROUND: Recently, linezolid-resistant staphylococci have become an emerging problem worldwide. Understanding the mechanisms of resistance, molecular epidemiology and transmission of linezolid-resistant CoNS in hospitals is very important. METHODS: The antimicrobial susceptibilities of all isolates were determined by the microdilution method. The resistance mechanisms and molecular characteristics of the strains were determined using whole-genome sequencing and PCR. RESULTS: All the strains were resistant to oxacillin and carried the mecA gene; 13 patients (36.1%) had prior linezolid exposure. Most S. epidermidis and S. hominis isolates were ST22 and ST1, respectively. MLST typing and evolutionary analysis indicated most linezolid-resistant CoNS strains were genetically related. In this study, we revealed that distinct CoNS strains have different mechanisms of linezolid resistance. Among ST22-type S. epidermidis, acquisition of the T2504A and C2534T mutations in the V domain of the 23 S rRNA gene, as well as mutations in the ribosomal proteins L3 (L101V, G152D, and D159Y) and L4 (N158S), were linked to the development of linezolid resistance. In S. cohnii isolates, cfr, S158Y and D159Y mutations in the ribosomal protein L3 were detected. Additionally, emergence of the G2576T mutation and the cfr gene were major causes of linezolid resistance in S. hominis isolates. The cfr gene, G2576T and C2104T mutations, M156T change in L3 protein, and I188S change in L4 protein were found in S. capitis isolates. CONCLUSION: The emergence of linezolid-resistant CoNS in the environment is concerning because it involves clonal dissemination and frequently coexists with various drug resistance mechanisms.

Integrative and Conjugative Elements and Prophage DNA as Carriers of Resistance Genes in Erysipelothrix rhusiopathiae Strains from Domestic Geese in Poland.

Goose erysipelas is a serious problem in waterfowl breeding in Poland. However, knowledge of the characteristics of Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, the antimicrobial susceptibility and serotypes of four E. rhusiopathiae strains from domestic geese were determined, and their whole-genome sequences (WGSs) were analyzed to detect resistance genes, integrative and conjugative elements (ICEs), and prophage DNA. Sequence type and the presence of resistance genes and transposons were compared with 363 publicly available E. rhusiopathiae strains, as well as 13 strains of other Erysipelothrix species. Four strains tested represented serotypes 2 and 5 and the MLST groups ST 4, 32, 242, and 243. Their assembled circular genomes ranged from 1.8 to 1.9 kb with a GC content of 36-37%; a small plasmid was detected in strain 1023. Strains 1023 and 267 were multidrug-resistant. The resistance genes detected in the genome of strain 1023 were erm47, tetM, and lsaE-lnuB-ant(6)-Ia-spw cluster, while strain 267 contained the tetM and ermB genes. Mutations in the gyrA gene were detected in both strains. The tetM gene was embedded in a Tn916-like transposon, which in strain 1023, together with the other resistance genes, was located on a large integrative and conjugative-like element of 130 kb designated as ICEEr1023. A minor integrative element of 74 kb was identified in strain 1012 (ICEEr1012). This work contributes to knowledge about the characteristics of E. rhusiopathiae bacteria and, for the first time, reveals the occurrence of erm47 and ermB resistance genes in strains of this species. Phage infection appears to be responsible for the introduction of the ermB gene into the genome of strain 267, while ICEs most likely play a key role in the spread of the other resistance genes identified in E. rhusiopathiae.

An Explainable Deep Learning Classifier of Bovine Mastitis Based on Whole-Genome Sequence Data-Circumventing the p >> n Problem.

The serious drawback underlying the biological annotation of whole-genome sequence data is the p >> n problem, which means that the number of polymorphic variants (p) is much larger than the number of available phenotypic records (n). We propose a way to circumvent the problem by combining a LASSO logistic regression with deep learning to classify cows as susceptible or resistant to mastitis, based on single nucleotide polymorphism (SNP) genotypes. Among several architectures, the one with 204,642 SNPs was selected as the best. This architecture was composed of two layers with, respectively, 7 and 46 units per layer implementing respective drop-out rates of 0.210 and 0.358. The classification of the test data resulted in AUC = 0.750, accuracy = 0.650, sensitivity = 0.600, and specificity = 0.700. Significant SNPs were selected based on the SHapley Additive exPlanation (SHAP). As a final result, one GO term related to the biological process and thirteen GO terms related to molecular function were significantly enriched in the gene set that corresponded to the significant SNPs. Our findings revealed that the optimal approach can correctly predict susceptibility or resistance status for approximately 65% of cows. Genes marked by the most significant SNPs are related to the immune response and protein synthesis.

Identification of novel single nucleotide variants in the drug resistance mechanism of Mycobacterium tuberculosis isolates by whole-genome analysis.

BACKGROUND: Tuberculosis (TB) represents a major global health challenge. Drug resistance in Mycobacterium tuberculosis (MTB) poses a substantial obstacle to effective TB treatment. Identifying genomic mutations in MTB isolates holds promise for unraveling the underlying mechanisms of drug resistance in this bacterium. METHODS: In this study, we investigated the roles of single nucleotide variants (SNVs) in MTB isolates resistant to four antibiotics (moxifloxacin, ofloxacin, amikacin, and capreomycin) through whole-genome analysis. We identified the drug-resistance-associated SNVs by comparing the genomes of MTB isolates with reference genomes using the MuMmer4 tool. RESULTS: We observed a strikingly high proportion (94.2%) of MTB isolates resistant to ofloxacin, underscoring the current prevalence of drug resistance in MTB. An average of 3529 SNVs were detected in a single ofloxacin-resistant isolate, indicating a mutation rate of approximately 0.08% under the selective pressure of ofloxacin exposure. We identified a set of 60 SNVs associated with extensively drug-resistant tuberculosis (XDR-TB), among which 42 SNVs were non-synonymous mutations located in the coding regions of nine key genes (ctpI, desA3, mce1R, moeB1, ndhA, PE_PGRS4, PPE18, rpsA, secF). Protein structure modeling revealed that SNVs of three genes (PE_PGRS4, desA3, secF) are close to the critical catalytic active sites in the three-dimensional structure of the coding proteins. CONCLUSION: This comprehensive study elucidates novel resistance mechanisms in MTB against antibiotics, paving the way for future design and development of anti-tuberculosis drugs.

Genomic insights into the diversity, virulence, and antimicrobial resistance of group B Streptococcus clinical isolates from Saudi Arabia.

Introduction: Detailed assessment of the population structure of group B Streptococcus (GBS) among adults is still lacking in Saudi Arabia. Here we characterized a representative collection of isolates from colonized and infected adults. Methods: GBS isolates (n=89) were sequenced by Illumina and screened for virulence and antimicrobial resistance determinants. Genetic diversity was assessed by single nucleotide polymorphisms and core-genome MLST analyses. Results: Genome sequences revealed 28 sequence types (STs) and nine distinct serotypes, including uncommon serotypes VII and VIII. Majority of these STs (n=76) belonged to the human-associated clonal complexes (CCs) CC1 (33.71%), CC19 (25.84%), CC17 (11.24%), CC10/CC12 (7.87%), and CC452 (6.74%). Major CCs exhibited intra-lineage serotype diversity, except for the hypervirulent CC17, which exclusively expressed serotype III. Virulence profiling revealed that nearly all isolates (94.38%) carried at least one of the four alpha family protein genes (i.e., alphaC, alp1, alp2/3, and rib), and 92.13% expressed one of the two serine-rich repeat surface proteins Srr1 or Srr2. In addition, most isolates harbored the pilus island (PI)-2a alone (15.73%) or in combination with PI-1 (62.92%), and those carrying PI-2b alone (10.11%) belonged to CC17. Phylogenetic analysis grouped the sequenced isolates according to CCs and further subdivided them along with their serotypes. Overall, isolates across all CC1 phylogenetic clusters expressed Srr1 and carried the PI-1 and PI-2a loci, but differed in genes encoding the alpha-like proteins. CC19 clusters were dominated by the III/rib/srr1/PI-1+PI-2a (43.48%, 10/23) and V/alp1/srr1/PI-1+PI-2a (34.78%, 8/23) lineages, whereas most CC17 isolates (90%, 9/10) had the same III/rib/srr2/P1-2b genetic background. Interestingly, genes encoding the CC17-specific adhesins HvgA and Srr2 were detected in phylogenetically distant isolates belonging to ST1212, suggesting that other highly virulent strains might be circulating within the species. Resistance to macrolides and/or lincosamides across all major CCs (n=48) was associated with the acquisition of erm(B) (62.5%, 30/48), erm(A) (27.1%, 13/48), lsa(C) (8.3%, 4/48), and mef(A) (2.1%, 1/48) genes, whereas resistance to tetracycline was mainly mediated by presence of tet(M) (64.18%, 43/67) and tet(O) (20.9%, 14/67) alone or in combination (13.43%, 9/67). Discussion: These findings underscore the necessity for more rigorous characterization of GBS isolates causing infections.

Complete Genome Sequencing, Annotation, and Mutational Profiling of the Novel Clade I Human Mpox Virus, Kamituga Strain.

INTRODUCTION: Human Mpox (formerly monkeypox) infection is an emerging zoonotic disease caused by the Mpox virus (MPXV). We describe the complete genome annotation, phylogeny, and mutational profile of a novel, sustained Clade I Mpox outbreak in the city of Kamituga in Eastern Democratic Republic of the Congo (DRC). METHODOLOGY: A cross-sectional, observational, cohort study was performed among patients of all ages admitted to the Kamituga Hospital with Mpox infection symptoms between late September 2023 and late January 2024. DNA was isolated from Mpox swabbed lesions and sequenced followed by phylogenetic analysis, genome annotation, and mutational profiling. RESULTS: We describe an ongoing Clade I Mpox outbreak in the city of Kamituga, South Kivu Province, Democratic Republic of Congo. Whole-genome sequencing of the viral RNA samples revealed, on average, 201.5 snps, 28 insertions, 81 deletions, 2 indels, 312.5 total variants, 158.3 amino acid changes, 81.66 intergenic variants, 72.16 synonymous mutations, 106 missense variants, 41.16 frameshift variants, and 3.33 inframe deletions across six samples. By assigning mutations at the proteome level for Kamituga MPXV sequences, we observed that seven proteins, namely, C9L (OPG047), I4L (OPG080), L6R (OPG105), A17L (OPG143), A25R (OPG151), A28L (OPG153), and B21R (OPG210) have emerged as hot spot mutations based on the consensuses inframe deletions, frameshift variants, synonymous variants, and amino acids substitutions. Based on the outcome of the annotation, we found a deletion of the D14L (OPG032) gene in all six samples. Following phylogenetic analysis and whole genome assembly, we determined that this cluster of Mpox infections is genetically distinct from previously reported Clade I outbreaks, and thus propose that the Kamituga Mpox outbreak represents a novel subgroup (subgroup VI) of Clade I MPXV. CONCLUSIONS: Here we report the complete viral genome for the ongoing Clade I Mpox Kamituga outbreak for the first time. This outbreak presents a distinct mutational profile from previously sequenced Clade I MPXV oubtreaks, suggesting that this cluster of infections is a novel subgroup (we term this subgroup VI). These findings underscore the need for ongoing vigilance and continued sequencing of novel Mpox threats in endemic regions.

Posibilities for use of whole genome sequencing (WGS) for the analysis of Streptococcus pneumoniae isolates.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive coccus causing both non-invasive and invasive infectious diseases. Pneumococcal diseases are vaccine preventable. Invasive pneumococcal diseases (IPD) meeting the international case definition are reported nationally and internationally and are subject to surveillance programmes in many countries, including the Czech Republic. An important part of IPD surveillance is the monitoring of causative serotypes and their frequency over time and in relation to ongoing vaccination programmes. In the world and in the Czech Republic, whole genome sequencing (WGS) is increasingly used for pneumococci, which allows for serotyping from sequencing data, precise analysis of their genetic relationships, and the study of genes present in their genome. Whole-genome sequencing enables the generation of reliable and internationally comparable data that can be easily shared. Sequencing data are analysed using bioinformatics tools that require knowledge in the field of natural sciences with an emphasis on genetics and expertise in bioinformatics. This publication presents some options for pneumococcal analysis, i.e., serotyping, multilocus sequence typing (MLST), ribosomal MLST (rMLST), core genome MLST (cgMLST), whole genome MLST (wgMLST), single nucleotide polymorphism (SNP) analysis, assignment to Global Pneumococcal Sequence Cluster (GPSC), and identification of virulence genes and antibiotic resistance genes. The WGS strategies and applications for Europe and WGS implementation in practice are presented. WGS analysis of pneumococci allows for improved IPD surveillance, thanks to molecular serotyping, more detailed typing, generation of internationally comparable data, and improved evaluation of the effectiveness of vaccination programmes.

Genetic Rare Variants Affecting Multiple Pathways in Japanese Patients with Palindromic Rheumatism.

Palindromic rheumatism (PR) is a type of cryptogenic paroxysmal arthritis. Several genes may be involved in PR pathogenesis; however, conducting comprehensive case-control genetic studies for PR poses challenges owing to its rarity as a disease. Moreover, case-control studies may overlook rare variants that occur infrequently but play a significant role in pathogenesis. This study aimed to identify disease-related genes in Japanese patients with PR using whole-genome sequencing (WGS) and rare-variant analysis. Genomic DNA was obtained from two familial cases and one sporadic case, and it was subjected to WGS. WGS data of 104 healthy individuals obtained from a public database were used as controls. We performed data analysis for rare variants on detected variants using SKAT-O, KBAC, and SKAT, and subsequently defined significant genes. Significant genes combined with variants shared between the cases were defined as disease-related genes. We also performed pathway analysis for disease-related genes using Reactome. We identified 2,695,244 variants shared between cases; after excluding polymorphisms and noise, 74,640 variants were detected. We identified 540 disease-related genes, including 1,893 variants. Furthermore, we identified 32 significant pathways. Our results indicate that the detected genes and pathways in this study may be involved in PR pathogenesis.

Whole-genome sequencing of Ganoderma boninense, the causal agent of basal stem rot disease in oil palm, via combined short- and long-read sequencing.

The hemibiotrophic Basidiomycete pathogen Ganoderma boninense (Gb) is the dominant causal agent of oil palm basal stem rot disease. Here, we report a complete chromosomal genome map of Gb using a combination of short-read Illumina and long-read Pacific Biosciences (PacBio) sequencing platforms combined with chromatin conformation capture data from the Chicago and Hi-C platforms. The genome was 55.87 Mb in length and assembled to a high contiguity (N50: 304.34 kb) of 12 chromosomes built from 112 scaffolds, with a total of only 4.34 Mb (~ 7.77%) remaining unplaced. The final assemblies were evaluated for completeness of the genome by using Benchmarking Universal Single Copy Orthologs (BUSCO) v4.1.4, and based on 4464 total BUSCO polyporales group searches, the assemblies yielded 4264 (95.52%) of the conserved orthologs as complete and only a few fragmented BUSCO of 42 (0.94%) as well as a missing BUSCO of 158 (3.53%). Genome annotation predicted a total of 21,074 coding genes, with a GC content ratio of 59.2%. The genome features were analyzed with different databases, which revealed 2471 Gene Ontology/GO (11.72%), 5418 KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthologous/KO (25.71%), 13,913 Cluster of Orthologous Groups of proteins/COG (66.02%), 60 ABC transporter (0.28%), 1049 Carbohydrate-Active Enzymes/CAZy (4.98%), 4005 pathogen-host interactions/PHI (19%), and 515 fungal transcription factor/FTFD (2.44%) genes. The results obtained in this study provide deep insight for further studies in the future.

Raw meat-based diet for pets: a neglected source of human exposure to Salmonella and pathogenic Escherichia coli clones carrying mcr, Portugal, September 2019 to January 2020.

BackgroundThe pet industry is expanding worldwide, particularly raw meat-based diets (RMBDs). There are concerns regarding the safety of RMBDs, especially their potential to spread clinically relevant antibiotic-resistant bacteria or zoonotic pathogens.AimWe aimed to investigate whether dog food, including RMBD, commercially available in Portugal can be a source of Salmonella and/or other Enterobacteriaceae strains resistant to last-line antibiotics such as colistin.MethodsFifty-five samples from 25 brands (21 international ones) of various dog food types from 12 suppliers were screened by standard cultural methods between September 2019 and January 2020. Isolates were characterised by phenotypic and genotypic methods, including whole genome sequencing and comparative genomics.ResultsOnly RMBD batches were contaminated, with 10 of 14 containing polyclonal multidrug-resistant (MDR) Escherichia coli and one MDR Salmonella. One turkey-based sample contained MDR Salmonella serotype 1,4,[5],12:i:- ST34/cgST142761 with similarity to human clinical isolates occurring worldwide. This Salmonella exhibited typical antibiotic resistance (bla TEM + strA-strB + sul2 + tet(B)) and metal tolerance profiles (pco + sil + ars) associated with the European epidemic clone. Two samples (turkey/veal) carried globally dispersed MDR E. coli (ST3997-complexST10/cgST95899 and ST297/cgST138377) with colistin resistance (minimum inhibitory concentration: 4 mg/L) and mcr-1 gene on IncX4 plasmids, which were identical to other IncX4 circulating worldwide.ConclusionSome RMBDs from European brands available in Portugal can be a vehicle for clinically relevant MDR Salmonella and pathogenic E. coli clones carrying genes encoding resistance to the last-line antibiotic colistin. Proactive actions within the One Health context, spanning regulatory, pet-food industry and consumer levels, are needed to mitigate these public health risks.

Increased susceptibility to Mycobacterium avium complex infection in miniature Schnauzer dogs caused by a codon deletion in CARD9.

Mammals are generally resistant to Mycobacterium avium complex (MAC) infections. We report here on a primary immunodeficiency disorder causing increased susceptibility to MAC infections in a canine breed. Adult Miniature Schnauzers developing progressive systemic MAC infections were related to a common founder, and pedigree analysis was consistent with an autosomal recessive trait. A genome-wide association study and homozygosity mapping using 8 infected, 9 non-infected relatives, and 160 control Miniature Schnauzers detected an associated region on chromosome 9. Whole genome sequencing of 2 MAC-infected dogs identified a codon deletion in the CARD9 gene (c.493_495del; p.Lys165del). Genotyping of Miniature Schnauzers revealed the presence of this mutant CARD9 allele worldwide, and all tested MAC-infected dogs were homozygous mutants. Peripheral blood mononuclear cells from a dog homozygous for the CARD9 variant exhibited a dysfunctional CARD9 protein with impaired TNF-α production upon stimulation with the fungal polysaccharide ß-glucan that activates the CARD9-coupled C-type lectin receptor, Dectin-1. While CARD9-deficient knockout mice are susceptible to experimental challenges by fungi and mycobacteria, Miniature Schnauzer dogs with systemic MAC susceptibility represent the first spontaneous animal model of CARD9 deficiency, which will help to further elucidate host defense mechanisms against mycobacteria and fungi and assess potential therapies for animals and humans.

Whole genome sequence analysis of shallot virus X from India reveals it to be a natural recombinant with positive selection pressure.

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.

Zoonotic bacterial and parasitic intestinal pathogens in foxes, raccoons and other predators from eastern Germany.

In this study, we investigated faecal specimens from legally hunted and road-killed red foxes, raccoons, raccoon dogs, badgers and martens in Germany for parasites and selected zoonotic bacteria. We found that Baylisascaris procyonis, a zoonotic parasite of raccoons, had spread to northeastern Germany, an area previously presumed to be free of this parasite. We detected various pathogenic bacterial species from the genera Listeria, Clostridium (including baratii), Yersinia and Salmonella, which were analysed using whole-genome sequencing. One isolate of Yersinia enterocolitica contained a virulence plasmid. The Salmonella Cholerasuis isolate encoded an aminoglycoside resistance gene and a parC point mutation, conferring resistance to ciprofloxacin. We also found tetracycline resistance genes in Paeniclostridium sordellii and Clostridium baratii. Phylogenetic analyses revealed that the isolates were polyclonal, indicating the absence of specific wildlife-adapted clones. Predators, which scavenge from various sources including human settlements, acquire and spread zoonotic pathogens. Therefore, their role should not be overlooked in the One Health context.

SARS-CoV-2 shedding, infectivity, and evolution in an immunocompromised adult patient.

This study aimed to provide further insight into the evolutionary dynamics of SARS-CoV-2 by analyzing the case of a 40-year-old man who had previously undergone autologous hematopoietic stem cell transplantation due to a diffuse large B-cell lymphoma. He developed a persistent SARS-CoV-2 infection lasting at least 218 days and did not manifest a humoral immune response to the virus during this follow-up period. Whole-genome sequencing and viral cultures confirmed a persistent infection with a replication-positive virus that had undergone genetic variation for at least 196 days after symptom onset.

Insights into the novel Enterococcus faecalis phage: A comprehensive genome analysis.

Enterococcus faecalis, a Gram-positive bacterium, poses a significant clinical challenge owing to its intrinsic resistance to a broad spectrum of antibiotics, warranting urgent exploration of innovative therapeutic strategies. This study investigated the viability of phage therapy as an alternative intervention for antibiotic-resistant E. faecalis, with a specific emphasis on the comprehensive genomic analysis of bacteriophage SAM-E.f 12. The investigation involved whole-genome sequencing of SAM-E.f 12 using Illumina technology, resulting in a robust dataset for detailed genomic characterization. Bioinformatics analyses were employed to predict genes and assign functional annotations. The bacteriophage SAM-E.f 12, which belongs to the Siphoviridae family, exhibited substantial potential, with a burst size of 5.7 PFU/infected cells and a latent period of 20 min. Host range determination experiments demonstrated its effectiveness against clinical E. faecalis strains, positioning SAM-E.f 12 as a precise therapeutic agent. Stability assays underscore resilience across diverse environmental conditions. This study provides a comprehensive understanding of SAM-E.f 12 genomic composition, lytic lifecycle parameters, and practical applications, particularly its efficacy in murine wound models. These results emphasize the promising role of phage therapy, specifically its targeted approach against antibiotic-resistant E. faecalis strains. The nuanced insights derived from this research will contribute to the ongoing pursuit of efficacious phage therapies and offer valuable implications for addressing the clinical challenges associated with E. faecalis infections.

First detection and characterization of mcr-1 colistin resistant E. coli from wild rat in Bangladesh.

Colistin resistance is a global concern warning for a one health approach to combat the challenge. Colistin resistant E. coli and their resistance determinants are widely distributed in the environment, and rats could be a potential source of these isolates and resistant determinants to a diverse environmental setting. This study was aimed to determine the presence of colistin resistant E. coli (CREC) in wild rats, their antimicrobial resistance (AMR) phenotypes, and genotypic analysis of mcr-1 CREC through whole genome sequencing (WGS). A total of 39 rats were examined and CREC was isolated from their fecal pellets onto MacConkey agar containing colistin sulfate (1 µg/ mL). AMR of the CREC was determined by disc diffusion and broth microdilution was employed to determine MIC to colistin sulfate. CREC were screened for mcr genes (mcr-1 to mcr-8) and phylogenetic grouping by PCR. Finally, WGS of one mcr-1 CREC was performed to explore its genetic characteristics especially resistomes and virulence determinants. 43.59% of the rats carried CREC with one (2.56%) of them carrying CREC with mcr-1 gene among the mcr genes examined. Examination of seventeen (17) isolates from the CREC positive rats (n = 17) revealed that majority of them belonging to the pathogenic phylogroup D (52.94%) and B2 (11.76%). 58.82% of the CREC were MDR on disc diffusion test. Shockingly, the mcr-1 CREC showed phenotypic resistance to 16 antimicrobials of 8 different classes and carried the ARGs in its genome. The mcr-1 gene was located on a 60 kb IncI2 plasmid. On the other hand, ARGs related to aminoglycosides, phenicols, sulfonamides, tetracyclines and trimethoprims were located on a 288 kb mega-plasmid separately. The mcr-1 CREC carried 58 virulence genes including genes related to adhesion, colonization, biofilm formation, hemolysis and immune-evasion. The isolate belonged to ST224 and closely related to E. coli from different sources including UPEC clinical isolates from human based on cgMLST analysis. The current research indicates that rats might be a possible source of CREC, and the presence of mcr-1 and other ARGs on plasmid increases the risk of ARGs spreading and endangering human health and other environmental components through this infamous pest.