Unraveling novel mutation patterns and morphological variations in two dalbavancin-resistant MRSA strains in Austria using whole genome sequencing and transmission electron microscopy.

BACKGROUND: The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) strains resistant to non-beta-lactam antimicrobials poses a significant challenge in treating severe MRSA bloodstream infections. This study explores resistance development and mechanisms in MRSA isolates, especially after the first dalbavancin-resistant MRSA strain in our hospital in 2016. METHODS: This study investigated 55 MRSA bloodstream isolates (02/2015-02/2021) from the University Hospital of the Medical University of Vienna, Austria. The MICs of dalbavancin, linezolid, and daptomycin were assessed. Two isolates (16-33 and 19-362) resistant to dalbavancin were analyzed via whole-genome sequencing, with morphology evaluated using transmission electron microscopy (TEM). RESULTS: S.aureus BSI strain 19-362 had two novel missense mutations (p.I515M and p.A606D) in the pbp2 gene. Isolate 16-33 had a 534 bp deletion in the DHH domain of GdpP and a SNV in pbp2 (p.G146R). Both strains had mutations in the rpoB gene, but at different positions. TEM revealed significantly thicker cell walls in 16-33 (p < 0.05) compared to 19-362 and dalbavancin-susceptible strains. None of the MRSA isolates showed resistance to linezolid or daptomycin. CONCLUSION: In light of increasing vancomycin resistance reports, continuous surveillance is essential to comprehend the molecular mechanisms of resistance in alternative MRSA treatment options. In this work, two novel missense mutations (p.I515M and p.A606D) in the pbp2 gene were newly identified as possible causes of dalbavancin resistance.

Unveiling the complexity of rifampicin drug susceptibility testing in Mycobacterium tuberculosis: comparative analysis with next-generation sequencing.

Introduction. The discordance between phenotypic and molecular methods of rifampicin (RIF) drug susceptibility testing (DST) in Mycobacterium tuberculosis poses a significant challenge, potentially resulting in misdiagnosis and inappropriate treatment.Hypothesis/gap statement. A comparison of RIF phenotypic and molecular methods for DST, including whole genome sequencing (WGS), may provide a better understanding of resistance mechanisms.Aim. This study aims to compare RIF DST in M. tuberculosis using two phenotypic and molecular methods including the GeneXpert RIF Assay (GX) and WGS for better understanding.Methodology. The study evaluated two phenotypic liquid medium methods [Lowenstein-Jensen (LJ) and Mycobacterium Growth Indicator Tube (MGIT)], one targeted molecular method (GX), and one WGS method. Moreover, mutational frequency in ponA1 and ponA2 was also screened in the current and previous RIF resistance M. tuberculosis genomic isolates to find their compensatory role.Results. A total of 25 RIF-resistant isolates, including nine from treatment failures and relapse cases with both discordant and concordant DST results on LJ, MGIT and GX, were subjected to WGS. The phenotypic DST results indicated that 11 isolates (44%) were susceptible on LJ and MGIT but resistant on GX. These isolates exhibited multiple mutations in rpoB, including Thr444>Ala, Leu430>Pro, Leu430>Arg, Asp435>Gly, His445>Asn and Asn438>Lys. Conversely, four isolates that were susceptible on GX and MGIT but resistant on LJ were wild type for rpoB in WGS. However, these isolates possessed several novel mutations in the PonA1 gene, including a 10 nt insertion and two nonsynonymous mutations (Ala394>Ser, Pro631>Ser), as well as one nonsynonymous mutation (Pro780>Arg) in PonA2. The discordance rate of RIF DST is higher on MGIT than on LJ and GX when compared to WGS. These discordances in the Delhi/CAS lineages were primarily associated with failure and relapse cases.Conclusion. The WGS of RIF resistance is relatively expensive, but it may be considered for isolates with discordant DST results on MGIT, LJ and GX to ensure accurate diagnosis and appropriate treatment options.

Assessment of the Safety and Exopolysaccharide Synthesis Capabilities of Bacillus amyloliquefaciens D189 Based on Complete Genome and Phenotype Analysis.

Exopolysaccharides (EPS) are natural macromolecular carbohydrates with good functional activity and physiological activities, which can be utilized as an emulsifier, viscosity enhancer, stabilizer, gelling agent, and water retention agent in a wide range of food products. In this study, the whole genome of Bacillus amyloliquefaciens D189, an EPS-producing bacteria, was sequenced. The result showed that D189 contains a single, circular chromosome of 3,963,356 bp with an average GC content of 45.74% and 3996 coding genes. The gene annotation results showed that D189 is a potentially safe strain and confirmed to be safe associated with hemolytic assay, and antibiotic resistance test. Meanwhile, D189 genome possessed 240 genes related to carbohydrate metabolism. More importantly, D189 could transport 9 sugars and contained a complete biosynthetic pathway for 8 nucleotide sugars. Based on the validation experiments, strain D189 could metabolize 8 sugars (glucose, sucrose, trehalose, fructose, cellobiose, maltose, mannitol, and N-acetyl-D-glucosamine) to produce EPS, with the highest yield of 1.212 g/L when sucrose was the carbon source. Therefore, the whole genome sequencing preliminarily elucidated the physiological mechanism of EPS, providing several pathways for engineering D189 to further enhance the yield of EPS.

Epidemiology and molecular evolution of GI.1 sapovirus in the recent era.

Sapovirus (SaV) infection is increasing worldwide. Herein, we provided evidence of a significant increase in SaV infection in Japan during 2010-2022, primarily due to the considerable (p = 0.0003) rise of the GI.1 genotype. Furthermore, we found that all major and minor SaV outbreaks in Japan, including the largest SaV outbreak in 2021-2022, were caused by the GI.1 genotype. Therefore, to get insight into the underlying molecular mechanism behind this rising trend of the SaV GI.1 type, we selected 15 SaV GI.1 outbreak strains for complete genome analysis through next-generation sequencing. Phylogenetically, our strains remained clustered in different branches in lineages I and II among the GI.1 genotype. We showed all amino acid (aa) substitutions in different open reading frames (ORFs) in these strains. Importantly, we have demonstrated that the strains involved in the largest SaV outbreak in Japan in 2021-2022 belonged to lineage II and possessed the third ORF. We have identified some unique aa mutations in these major outbreak strains in the NS1 and NS6-NS7 regions that are thought to be associated with viral pathogenicity, cell tropism, and epidemiological competence. Thus, in addition to enriching the database of SaV's complete sequences, this study provides insights into its important mutations.

First Application of Whole Genome Sequencing in Myelinated Retinal Nerve Fibers (MRNF).

Genetic features are currently unknown in myelinated retinal nerve fibers (MRNF). For a 20-year-old asymptomatic female with unilateral MRNF, we performed whole genome sequencing (WGS) by standard workflow protocol to produce contiguous long-read sequences with Illumina DNA PCR-Free Prep. After tagmentation, libraries were sequenced on separate runs via NovaSeq 6000 platform at 2 x 150bp read length. Gene variants included rs2248799, rs2672589, rs7555070, rs247616_T and rs2043085_C all associated with an increased macular degeneration risk, and seven novel variants of uncertain significance. For optic disc enlargement, variants rs9988687_A, rs11079419_T, rs6787363 and rs10862708_A suggested an increased risk for this condition. In contrast, modeling revealed retinal detachment risk was reduced by variants identified at rs9651980_T, rs4373767_T, and rs7940691_T which were among five other previously unreported variants. WGS data placed proband at the 66th and 64th percentiles for disc anomaly and retinal detachment risk, respectively. Additionally, risk determined from 16 loci associated with age-related macular degeneration found the patient to be at the 18th percentile for this diagnosis (i.e., below average genetic predisposition). Fundoscopic findings showed mean RNFL thickness was lower with MRNF (77 OS vs. 96?m OD) and RNFL symmetry was impaired (43 %) but stable between 2020 and 2023. Rim area and cup volume were also substantially different (2.33 OS vs. 1.34mm2 OD, and 0.001 OS vs. 0.151mm3 OD, respectively). As the first known evaluation of MRNF via WGS, these data reveal a mixed picture with variants associated with different risks for potentially related ocular pathologies. In addition, we identify multiple new variants of unknown significance. Factors affecting gene expression in MRNF require further study. Key words: Whole genome sequencing, Retina, Myelination, Anatomy, Gene variants.

Mutations in the efflux regulator gene oqxR provide a simple genetic switch for antimicrobial resistance in Klebsiella pneumoniae.

Klebsiella pneumoniae is a pathogen of major concern in the global rise of antimicrobial resistance and has been implicated as a reservoir for the transfer of resistance genes between species. The upregulation of efflux pumps is a particularly concerning mechanism of resistance acquisition as, in many instances, a single point mutation can simultaneously provide resistance to a range of antimicrobials and biocides. The current study investigated mutations in oqxR, which encodes a negative regulator of the RND-family efflux pump genes, oqxAB, natively found in the chromosome of K. pneumoniae. Resistant mutants in four K. pneumoniae strains (KP6870155, NTUH-K2044, SGH10, and ATCC43816) were selected from single exposures to 30 µg/mL chloramphenicol and 12 mutants were selected for whole genome sequencing to identify mutations associated with resistance. Resistant mutants generated by single exposures to chloramphenicol, tetracycline, or ciprofloxacin at ≥4 X MIC were replica plated onto all three antibiotics to observe simultaneous cross-resistance to all compounds, indicative of a multidrug resistance phenotype. A variety of novel mutations, including single point mutations, deletions, and insertions, were found to disrupt oqxR leading to significant and simultaneous increases in resistance to chloramphenicol, tetracycline, and ciprofloxacin. The oqxAB-oqxR locus has been mobilized and dispersed on plasmids in many Enterobacteriaceae species and the diversity of these loci was examined to evaluate the evolutionary pressures acting on these genes. Comparison of the promoter regions of oqxR in plasmid-borne copies of the oqxR-oqxAB operon indicated that some constructs may produce truncated versions of the oqxR transcript, which may impact on oqxAB regulation and expression. In some instances, co-carriage of chromosomal and plasmid encoded oqxAB-oqxR was found in K. pneumoniae, implying that there is selective pressure to maintain and expand the efflux pump. Given that OqxR is a repressor of oqxAB, any mutation affecting its expression or function can lead to multidrug resistance. This is in contrast to antibiotic target site mutations that must occur in limited sequence space to be effective and not impact the fitness of the cell. Therefore, oqxR may act as a simple genetic switch to facilitate resistance via OqxAB mediated efflux.

Genomic and transcriptomic landscape to decipher the genetic basis of hyperpigmentation in Lanping black-boned sheep (Ovis aries).

BACKGROUND: Lanping black-boned sheep (LPB) represent a distinctive mammalian species characterized by hyperpigmentation, resulting in black bone and muscle features, in contrast to their conventional counterparts exhibiting red muscle and white bone. The genetic basis underlying LPB hyperpigmentation has remained enigmatic. METHODS: In this study, we conducted whole-genome sequencing of 100 LPB and 50 Lanping normal sheep (LPN), and integrated this data with 421 sequenced datasets from wild and domestic sheep, shedding light on the genetic backdrop and genomic variations associated with LPB. Furthermore, we performed comparative RNA-Seq analysis using liver sample to pinpoint genes implicated in the pigmentation process. We generated a comprehensive dataset comprising 97,944,357 SNPs from 571 sheep, facilitating an in-depth exploration of genetic factors. RESULTS: Population genetic structure analysis revealed that the LPB breed traces its origin back to LPN, having evolved into a distinct breed. The integration of positively selected genes with differentially expressed genes identified two candidates, ERBB4 and ROR1, potentially linked to LPB hyperpigmentation. Comparative analysis of ERBB4 and ROR1 mRNA relative expression levels in liver, spleen, and kidney tissues of LPB, in comparison to Diqing sheep, revealed significant upregulation, except for ERBB4 in the liver. Gene expression heatmaps further underscored marked allelic frequency disparities in different populations. CONCLUSION: Our findings establish the evolutionary lineage of the LPB breed from LPN and underscore the involvement of ERBB4 and ROR1 genes in melanin synthesis. These results enhance our comprehension of the molecular basis of hyperpigmentation and contribute to a more comprehensive depiction of sheep diversity.

Genomic portraits of methicillin-resistant staphylococci (MRS) from food fish unveiled the genes associated with staphylococcal food poisoning (SFP), virulence and antimicrobial resistance.

BACKGROUND: Characteristics of non-clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) especially from fishery environment are poorly understood. This research, in addition to comprehensive characterisation, sought to delineate the genetic relatedness between the MRSA strains originating from clinical as well as non-clinical settings. Out of 39 methicillin-resistant staphylococcal isolates from 197 fish samples, 6 (Three each of methicillin-resistant S. haemolyticus (MRSH) and MRSA) with distinct resistance profiles were selected for whole-genome sequencing. Using respective bioinformatics tools, MRSA genomes were comprehensively characterized for resistome, virulomes, molecular epidemiology and phylogenetic analysis. Simultaneously, MRSH genomes were specifically examined to characterize antimicrobial resistance genes (ARGs), owing to the fact that MRSH is often recognized as a reservoir for resistance determinants. RESULTS: Three MRSA clones identified in this study include ST672-IVd/t13599 (sequence type-SCCmec type/spa type), ST88-V/t2526, and ST672-IVa/t1309. Though, the isolates were phenotypically vancomycin-sensitive, five of the six genomes carried vancomycin resistance genes including the VanT (VanG cluster) or VanY (VanM cluster). Among the three MRSA, only one harbored the gene encoding Panton-Valentine Leukocidin (PVL) toxin, while staphylococcal enterotoxin (SEs) genes such as sea and seb, associated with staphylococcal food poisoning were identified in two other MRSA. Genomes of MRSH carried a composite of type V staphylococcal cassette chromosome mec (SCCmec) elements (5C2 & 5). This finding may be explained by the inversion and recombination events that may facilitate the integration of type V elements to the SCC elements of S. aureus with a methicillin-susceptible phenotype. Phylogenetically, MRSA from a non-clinical setting displayed a considerable relatedness to that from clinical settings. CONCLUSION: This study highlights the genetic diversity and resistance profiles of MRSA and MRSH, with non-clinical MRSA showing notable relatedness to clinical strains. Future research should explore resistance gene transfer mechanisms and environmental reservoirs to better manage MRSA spread.

Development and validation of a 66K SNP array for the hard clam (Mercenaria mercenaria).

BACKGROUND: The hard clam (Mercenaria mercenaria), a marine bivalve distributed along the U.S. eastern seaboard, supports a significant shellfish industry. Overharvest in the 1970s and 1980s led to a reduction in landings. While the transition of industry from wild harvest to aquaculture since that time has enhanced production, it has also exacerbated challenges such as disease outbreaks. In this study, we developed and validated a 66K SNP array designed to advance genetic studies and improve breeding programs in the hard clam, focusing particularly on the development of markers that could be useful in understanding disease resistance and environmental adaptability. RESULTS: Whole-genome resequencing of 84 individual clam samples and 277 pooled clam libraries yielded over 305 million SNPs, which were filtered down to a set of 370,456 SNPs that were used as input for the design of a 66K SNP array. This medium-density array features 66,543 probes targeting coding and non-coding regions, including 70 mitochondrial SNPs, to capture the extensive genetic diversity within the species. The SNPs were distributed evenly throughout the clam genome, with an average interval of 25,641 bp between SNPs. The array incorporates markers for detecting the clam pathogen Mucochytrium quahogii (formerly QPX), enhancing its utility in disease management. Performance evaluation on 1,904 samples demonstrated a 72.7% pass rate with stringent quality control. Concordance testing affirmed the array's repeatability, with an average agreement of allele calls of 99.64% across multiple tissue types, highlighting its reliability. The tissue-specific analysis demonstrated that some tissue types yield better genotyping results than others. Importantly, the array, including its embedded mitochondrial markers, effectively elucidated complex genetic relationships across different clam groups, both wild populations and aquacultured stocks, showcasing its utility for detailed population genetics studies. CONCLUSIONS: The 66K SNP array is a powerful and robust genotyping tool that offers unprecedented insights into the species' genomic architecture and population dynamics and that can greatly facilitate hard clam selective breeding. It represents an important resource that has the potential to transform clam aquaculture, thereby promoting industry sustainability and ecological and economic resilience.

Pan-genome analysis reveals novel chromosomal markers for multiplex PCR-based specific detection of Bacillus anthracis.

BACKGROUND: Bacillus anthracis is a highly pathogenic bacterium that can cause lethal infection in animals and humans, making it a significant concern as a pathogen and biological agent. Consequently, accurate diagnosis of B. anthracis is critically important for public health. However, the identification of specific marker genes encoded in the B. anthracis chromosome is challenging due to the genetic similarity it shares with B. cereus and B. thuringiensis. METHODS: The complete genomes of B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis were de novo annotated with Prokka, and these annotations were used by Roary to produce the pan-genome. B. anthracis exclusive genes were identified by Perl script, and their specificity was examined by nucleotide BLAST search. A local BLAST alignment was performed to confirm the presence of the identified genes across various B. anthracis strains. Multiplex polymerase chain reactions (PCR) were established based on the identified genes. RESULT: The distribution of genes among 151 whole-genome sequences exhibited three distinct major patterns, depending on the bacterial species and strains. Further comparative analysis between the three groups uncovered thirty chromosome-encoded genes exclusively present in B. anthracis strains. Of these, twenty were found in known lambda prophage regions, and ten were in previously undefined region of the chromosome. We established three distinct multiplex PCRs for the specific detection of B. anthracis by utilizing three of the identified genes, BA1698, BA5354, and BA5361. CONCLUSION: The study identified thirty chromosome-encoded genes specific to B. anthracis, encompassing previously described genes in known lambda prophage regions and nine newly discovered genes from an undefined gene region to the best of our knowledge. Three multiplex PCR assays offer an accurate and reliable alternative method for detecting B. anthracis. Furthermore, these genetic markers have value in anthrax vaccine development, and understanding the pathogenicity of B. anthracis.

Genomic approach to determine sources of neonatal Staphylococcus aureus infection from carriage in the Gambia.

Staphylococcus aureus is a major cause of neonatal infections in various anatomical sites, resulting in high morbidity and mortality in The Gambia. These clinical infections are often preceded by nasal carriage of S. aureus, a known risk factor. To determine whether potential sources of newborn S. aureus infections were from carriage, and to characterize S. aureus present in different anatomical sites (blood, ear, eye, umbilical cord, skin, pus, oropharynx, breast milk and vagina), we performed whole-genome sequencing of 172 isolates from clinical sites as well as from healthy and unhealthy carriage. A random selection of mothers (n = 90) and newborns (n = 42) participating in a clinical trial and testing positive for S. aureus were considered for this study. Sequence data were analyzed to determine S. aureus multilocus sequence types and selected antimicrobial and virulence gene profiles. Our findings revealed that in The Gambia, ST15 is the dominant sequence type associated with both carriage and clinical infection. In addition, S. aureus isolates causing clinical infection among neonates were genetically similar to those colonizing their oropharynx, and the different anatomical sites were not found to be uniquely colonized by S. aureus of a single genomic profile. Furthermore, while S. aureus associated with clinical infection had similar antimicrobial resistance gene profiles to carriage isolates, only hemolysin and adhesive factor virulence genes were significantly higher among clinical isolates. In conclusion, this study confirmed S. aureus oropharyngeal colonization among neonates as a potential source of clinical infection in The Gambia. Hence, interventions aiming to reduce neonatal clinical infections in The Gambia should consider decreasing oropharyngeal S. aureus carriage.Trial registration The trial was registered at ClinicalTrials.gov NCT03199547.

Genomic instability in congenital lung malformations in children.

PURPOSE: To study the biological relationship between congenital lung malformations (CLMs) and malignancy. METHODS: Biopsies of 12 CPAMs, 6 intralobar sequestrations and 2 extralobar sequestrations were analyzed through whole-genome sequencing. Blood samples from 10 patients were used to confirm or exclude somatic mosaicism. Putative somatic Single Nucleotide Variants (SNVs) were called for each malformed sample with a Panel of Normals built with control DNA samples extracted from blood. The variants were subsequently confirmed by Sanger sequencing and searched, whenever possible, in the blood samples of patients. RESULTS: All CLMs but one presented a signature of genomic instability by means of multiple clusters of cells with gene mutations. Seven tumor transformation-related SNVs were detected in 6/20 congenital lung malformations. Four very rare in the general population SNVs were found in a region previously linked to lung cancer in 5p15.33, upstream of TERT oncogene. Furthermore, we identified missense genetic variants, whose tumorigenic role is well known, in the RET, FANCA and MET genes. CONCLUSIONS: Genomic instability in 95% of CLMs and genetic variants linked to tumor development in 30% of them, regardless of histopathology, are predisposing factors to malignancy, that combined with exposure to carcinogens, might trigger the development of malignancy and explain the association between CLMs and lung cancer.

Transfer of Thermobacterium salinum Chen et al. 2023 to the genus Luteirhabdus Ren et al. 2022 as Luteirhabdus salina comb. nov.

The 16S rRNA gene of Thermobacterium salinum TK19130T had the highest sequence similarity to that of Luteirhabdus pelagi A3-108T (99.7%). Phylogeny of 16S rRNA gene and whole genome sequences indicated that T. salinum TK19130T and L. pelagi A3-108T are closely related, and represented an independent clade. Whole genome comparisons showed that T. salinum TK19130T and L. pelagi A3-108T shared average amino acid identity of 95.3%, indicating they could be merged into the same genus. The digital DNA-DNA hybridization and average nucleotide identity values between T. salinum TK19130T and L. pelagi A3-108T were 52.5 and 93.3%, respectively. These values were below the recommended threshold values of prokaryotic species delineation. Thus, based on the principle of priority, we proposed the transfer of Thermobacterium salinum Chen et al. 2023 to the genus Luteirhabdus Ren et al. 2022 as Luteirhabdus salina comb. nov.

Multi-omics and clustering analyses reveal the mechanisms underlying unmet needs for patients with lung adenocarcinoma and identify potential therapeutic targets.

BACKGROUND: The cancer genome contains several driver mutations. However, in some cases, no known drivers have been identified; these remaining areas of unmet needs, leading to limited progress in cancer therapy. Whole-genome sequencing (WGS) can identify non-coding alterations associated with the disease. Consequently, exploration of non-coding regions using WGS and other omics data such as ChIP-sequencing (ChIP-seq) to discern novel alterations and mechanisms related to tumorigenesis have been attractive these days. METHODS: Integrated multi-omics analyses, including WGS, ChIP-seq, DNA methylation, and RNA-sequencing (RNA-seq), were conducted on samples from patients with non-clinically actionable genetic alterations (non-CAGAs) in lung adenocarcinoma (LUAD). Second-level cluster analysis was performed to reinforce the correlations associated with patient survival, as identified by RNA-seq. Subsequent differential gene expression analysis was performed to identify potential druggable targets. RESULTS: Differences in H3K27ac marks in non-CAGAs LUAD were found and confirmed by analyzing RNA-seq data, in which mastermind-like transcriptional coactivator 2 (MAML2) was suppressed. The down-regulated genes whose expression was correlated to MAML2 expression were associated with patient prognosis. WGS analysis revealed somatic mutations associated with the H3K27ac marks in the MAML2 region and high levels of DNA methylation in MAML2 were observed in tumor samples. The second-level cluster analysis enabled patient stratification and subsequent analyses identified potential therapeutic target genes and treatment options. CONCLUSIONS: We overcome the persistent challenges of identifying alterations or driver mutations in coding regions related to tumorigenesis through a novel approach combining multi-omics data with clinical information to reveal the molecular mechanisms underlying non-CAGAs LUAD, stratify patients to improve patient prognosis, and identify potential therapeutic targets. This approach may be applicable to studies of other cancers with unmet needs.

Evaluating the accuracy of the MBT Lipid Xtract Kit for assessing colistin resistance in comparison to broth microdilution.

Colistin resistance testing methods such as broth microdilution (BMD) are time-consuming and labour intensive for clinical laboratories. MBT Lipid Xtract Kit on MALDI Biotyper Sirius System (Bruker, Billerica, MA, USA) utilizes lipidomic analysis to identify specific cell wall modifications associated with colistin resistance. We compared MBT to BMD (ComASP Colistin, Liofilchem) across 36 Gram-negative isolates (non-resistant MIC ≤2 µg ml-1, resistant MIC ≥4 µg ml-1). All samples were tested twice on MBT with discrepant results repeated before assessing categorical agreement between MBT and BMD. 44.4% (16/36) of isolates were colistin resistant via BMD. MBT Lipid Xtract had 80.6% agreement (29/36) with BMD, with 5/7 discrepancies corrected to match upon repeat testing. There was 100% agreement for Escherichia coli isolates (n=16). The whole-genome sequencing was completed on the two discrepant Klebsiella pneumoniae isolates, with variants within colistin resistance-associated loci identified (MIC 0.5 µg ml-1: arnC S30T, pmrB T246A, lapB N212T, lpxM S253G, crrB Q287K and MIC >16 µg ml-1: arnC S30T, pmrB R90insRN, pmrB T246A, pmrA E57G, lpxM S253G). Further evaluation, particularly for non-E. coli, of MBT is required prior to implementation in clinical laboratories.

Surprising diversity of new plasmids in bacteria isolated from hemorrhoid patients.

Background: Hemorrhoids are common conditions at or around the anus, to which numerous people suffer worldwide. Previous research has suggested that microbes may play a role in the development of hemorrhoids, and the origins of these microbes have been preliminarily investigated. However, no detailed research on the microbes related to hemorrhoid patients has been conducted. This work aims to provide an initial investigation into the microbes related to hemorrhoid patients with high quality whole genome sequencing. Methods: Forty-nine bacterial strains were isolated from seven hemorrhoid patients. Third-generation nanopore sequencing was performed to obtain high quality whole genome sequences. The presence of plasmids, particularly new plasmids, along with antibiotic resistance genes, was investigated for these strains. Phylogenetic analysis and genome comparisons were performed. Results: Out of the 31 plasmids found in the strains, 15 new plasmids that have not been observed previously were discovered. Further structural analysis revealed new multidrug-resistant conjugative plasmids, virulent plasmids, and small, high-copy mobile plasmids that may play significant functional roles. These plasmids were found to harbor numerous integrases, transposases, and recombinases, suggesting their ability to quickly obtain genes to change functions. Analysis of antibiotic resistance genes revealed the presence of antibiotic resistant-integrons. Together with the surprising number of new plasmids identified, as well as the finding of transmission and modification events for plasmids in this work, we came to the suggestion that plasmids play a major role in genetic plasticity. Conclusion: This study reveals that the diversity of plasmids in human-associated microbes has been underestimated. With the decreasing cost of whole-genome sequencing, monitoring plasmids deserves increased attention in future surveillance efforts.

Complete genome sequence of grapevine yellow speckle viroid 3, a novel apscaviroid infecting grapevine, characterized by high-throughput sequencing.

A novel grapevine viroid was discovered in an asymptomatic grapevine of Indian rootstocks. The whole genome sequence of the viroid (370 nt) was determined by high-throughput sequencing as well as RT-PCR followed by cloning and Sanger sequencing. The terminal conserved region (TCR), central conserved region (CCR) upper strand, and CCR lower strand are conserved regions found in the viroid that are unique to the members of the genus Apscaviroid. Based on our findings and the demarcation criteria for viroids, the novel viroid, which we have tentatively named "grapevine yellow speckle viroid 3" is a putative new member of the genus Apscaviroid.

Large-scale analysis of whole genome sequencing data from formalin-fixed paraffin-embedded cancer specimens demonstrates preservation of clinical utility.

Whole genome sequencing (WGS) provides comprehensive, individualised cancer genomic information. However, routine tumour biopsies are formalin-fixed and paraffin-embedded (FFPE), damaging DNA, historically limiting their use in WGS. Here we analyse FFPE cancer WGS datasets from England's 100,000 Genomes Project, comparing 578 FFPE samples with 11,014 fresh frozen (FF) samples across multiple tumour types. We use an approach that characterises rather than discards artefacts. We identify three artefactual signatures, including one known (SBS57) and two previously uncharacterised (SBS FFPE, ID FFPE), and develop an "FFPEImpact" score that quantifies sample artefacts. Despite inferior sequencing quality, FFPE-derived data identifies clinically-actionable variants, mutational signatures and permits algorithmic stratification. Matched FF/FFPE validation cohorts shows good concordance while acknowledging SBS, ID and copy-number artefacts. While FF-derived WGS data remains the gold standard, FFPE-samples can be used for WGS if required, using analytical advancements developed here, potentially democratising whole cancer genomics to many.

Virulence factors, antibiotic susceptibility and sequence type distribution of hospital-associated Clostridioides difficile isolates in Israel, 2020-2022.

Biofilm formation and toxin production are some of the virulence factors of Clostridioides difficile (C. difficile), which causes hospital-acquired C. difficile infection (HA-CDI). This work investigated the prevalence and distribution of different strains recovered from HA-CDI patients hospitalized in 4 medical centres across Israel, and characterized strains' virulence factors and antibiotic susceptibility. One-hundred and eighty-eight faecal samples were collected. C. difficile 's toxins were detected by the CerTest Clostridium difficile GDH + Toxin A + B combo card test kit. Toxin loci PaLoc and PaCdt were detected by whole-genome sequencing (WGS). Multi-locus sequence typing (MLST) was performed to classify strains. Biofilm production was assessed by crystal violet. Antibiotic susceptibility was determined using Etest. Fidaxomicin susceptibility was tested via agar dilution. Sequence type (ST) 42 was the most (13.8%) common strain. All strains harboured the 2 toxins genes; 6.9% had the binary toxin. Most isolates were susceptible to metronidazole (98.9%) and vancomycin (99.5%). Eleven (5.85%) isolates were fidaxomicin-resistant. Biofilm production capacity was associated with ST (p < 0.001). In conclusion, a broad variety of C. difficile strains circulate in Israel's medical centres. Further studies are needed to explore the differences and their contribution to HA-CDI epidemiology.

Long-term persistence of diverse clones shapes the transmission landscape of invasive Listeria monocytogenes.

BACKGROUND: The foodborne bacterium Listeria monocytogenes (Lm) causes a range of diseases, from mild gastroenteritis to invasive infections that have high fatality rate in vulnerable individuals. Understanding the population genomic structure of invasive Lm is critical to informing public health interventions and infection control policies that will be most effective especially in local and regional communities. METHODS: We sequenced the whole draft genomes of 936 Lm isolates from human clinical samples obtained in a two-decade active surveillance program across 58 counties in New York State, USA. Samples came mostly from blood and cerebrospinal fluid. We characterized the phylogenetic relationships, population structure, antimicrobial resistance genes, virulence genes, and mobile genetic elements. RESULTS: The population is genetically heterogenous, consisting of lineages I-IV, 89 clonal complexes, 200 sequence types, and six known serogroups. In addition to intrinsic antimicrobial resistance genes (fosX, lin, norB, and sul), other resistance genes tetM, tetS, ermG, msrD, and mefA were sparsely distributed in the population. Within each lineage, we identified clusters of isolates with ≤ 20 single nucleotide polymorphisms in the core genome alignment. These clusters may represent isolates that share a most recent common ancestor, e.g., they are derived from the same contamination source or demonstrate evidence of transmission or outbreak. We identified 38 epidemiologically linked clusters of isolates, confirming eight previously reported disease outbreaks and the discovery of cryptic outbreaks and undetected chains of transmission, even in the rarely reported Lm lineage III (ST3171). The presence of animal-associated lineages III and IV may suggest a possible spillover of animal-restricted strains to humans. Many transmissible clones persisted over several years and traversed distant sites across the state. CONCLUSIONS: Our findings revealed the bacterial determinants of invasive listeriosis, driven mainly by the diversity of locally circulating lineages, intrinsic and mobile antimicrobial resistance and virulence genes, and persistence across geographical and temporal scales. Our findings will inform public health efforts to reduce the burden of invasive listeriosis, including the design of food safety measures, source traceback, and outbreak detection.