Discovery of molecular mechanisms of primary osteoporosis development is fundamental to understand the pathogenesis of musculoskeletal diseases in general and for identifying key links in the genetic and epigenetic regulation of bone remodelling genes. The number of identified molecular genetic markers for osteoporosis is increasing but there is a need to describe their functional interactions. These interactions have been determined to be associated with the control of expression of a number of transcription factors and the differentiation of mesenchymal stem cells through the pathway of osteoblastogenesis or adipogenesis, and monocytic precursors through the pathway of osteoclastogenesis. The results of epigenetic studies have significantly increased the understanding of the role of post-translational modifications of histones, DNA methylation and RNA interference in the osteoporosis pathogenesis and in bone remodelling. However, the knowledge should be systematised and generalised according to the results of research on the role of epigenetic modifiers in the development of osteoporosis, and the influence of each epigenetic mechanism on the individual links of bone remodelling during ontogenesis of humans in general, including the elderly, should be described. Understanding which mechanisms and systems are involved in the development of this nosology is of interest for the development of targeted therapies, as the possibility of using microRNAs to regulate genes is now being considered. Systematisation of these data is important to investigate the differences in epigenetic marker arrays by race and ethnicity. The review article analyses references to relevant reviews and original articles, classifies information on current advances in the study of epigenetic mechanisms in osteoporosis and reviews the results of studies of epigenetic mechanisms on individual links of bone remodelling. Primary osteoporosis (OP) is an age-associated disease ofmultifactorial aetiology, which is based on a violation ofthe balance of bone remodelling, leading to a decrease inthe level of bone mineral density (BMD) and a violationof the structure of bone microarchitectonics, which resultin the appearance of an incorrect spatial structure of thespongy and cortical bone . Bone massis reduced to 26 % in individuals predisposed to OP. Inroughly 80 % of women, the mineral content in the spinefalls below the threshold value at the age of 60–70 years,and at 85 years – in more than 90 % .According to statistics, the frequency of fractures in womenis 33 %, in men, 20 % .Until recently, OP was diagnosed only by secondarysigns, such as low height and bone pain . In 1940, the American endocrinologist F. Albright,describing postmenopausal osteoporosis, suggestedthat it developed due to estrogen deficiency . On this basis, clinicians developed the now obsoleteconcept of two forms of OP, one associated withestrogen deficiency during menopause and the other withcalcium deficiency and skeletal ageing, both of which arecharacteristic of both sexes .Current evidence defines OP as a musculoskeletal diseaseassociated with profound metabolic changes not only inbone, but also in whole-body homeostasis: micro-nutrientand endocrine dysregulation, as well as a complex interactionof genetic, endogenous and environmental factors thatcontribute to a complex disease phenotype . Risk factors with regards to OP are assumedto be the female sex, being of the Caucasoid or Mongoloidrace , early menopause, old age, familyhistory, insufficient insolation, comorbidities with impairedbone matrix micronutrient absorption, smoking, alcoholabuse, sedentary lifestyle, taking hormonal drugs andrheumatoid arthritis. DNA testing has not been introducedinto diagnostic practice because significant populationdifferences in the frequency distribution of risk markersprevent the development of universal test systems, despitethe existence of significant and validated genetic markersfor OP .The first multicentre molecular genetic studies basedon the genome-wide association search (GWAS) methodconfirmed that OP is associated with genes for local andsystemic regulation of bone cell function. The lion’s shareof risk markers, specifically, single-nucleotide polymorphisms,have been identified not in exons but in intronsand promoters of regulatory genes, transcription factor,receptor (ESR2) and growth factor (FGF2) genes .Certain markers have been identified in non-coding RNA(ncRNA) sequences, including microRNA . Significant levels of associationswith fractures have been identified among the genesof systemic and local regulators ,transmembrane receptors, as well as WNT signalling genes,nuclear transcription factors  and enzymesthat produce or inactivate local bone regulators .Bioinformatics studies have concluded that the main enrichmentpathways by means of the functional affiliation ofgenes associated with histone modifications and microRNApatterns are significantly associated with the regulation ofRUNX2, FGF2 and SOX9 transcription factors. Moreover,they are also associated with the regulation of mesenchymalstem cell (MSC) differentiation .Thus, particular epigenetic factors in the pathogenesis ofosteoporosis have been identified. However, at present, it ismore interesting to consider the molecular pathogenesis ofOP in terms of individual functional links in the regulationof bone remodelling, in particular how RANK/RANKL/OPG, WNT, RUNX2 transcription factor and others areepigenetically regulated.Controlled differentiation of osteoblast and osteoclast precursors(mononuclear phagocytes) is necessary to maintainthe balance of bone remodelling .Osteoclast activity is primarily regulated by the RANK/RANKL/OPG (receptor-activator nuclear factor kβ/RANKligand/osteoprotegerin) system. RANKL is produced byosteoblasts and its binding to RANK on the osteoclast surface activates the expression of osteoclastogenic genes.The role of gene polymorphisms of this system in increasedrisk of fracture and the formation of low BMDhas previously been shown . Inthe RANKL gene sequence, two CpG sites were detected:one in the upstream sequence with 18 CpG sites, localizedat a distance of 14,415 pairs of nucleotides (bp) from thetranscription start site of the TSS I major isoform and onein the downstream sequence with 59 CpG sites, whichstarts at 260 and ends at 615 pairs of nucleotides of theTSS I isoform. In the OPG gene sequence, one island of56 sites spanning from –402 to +850 nucleotide pairs fromthe transcription site of the TSS isoform was observed. In , a groupof patients from Guangzhou Medical University Hospitalwith osteoporotic fractures had significantly higher RANKLgene mRNA levels from femoral bone cells compared tocontrols, with downregulated methylation status .Dysregulation of the expression of these genes is knownto be a key link in the development of steroid-inducedosteonecrosis of the femoral head and was noted to be associatedwith increased DNA methylation levels of OPGand RANK genes and with decreased levels in the RANKLgene . Previously, J. Delgado-Calle et al.(2012) performed a comparative analysis of the expressionlevels and methylation profile of the RANKL and OPGgenes in bone tissue samples from patients with osteoporoticfemoral neck fractures and osteoarthritis (OA) ofthe hip joint, which obtained remarkable results. RANKLexpression levels were significantly higher in patientswith fractures ( p = 0.012), while no significant changes inOPG gene expression levels were observed. The ratio ofRANKL ligand to OPG was higher in bone samples withOP . Differentialmethylation analysis revealed that the upstream promoterregion of the RANKL gene was strongly methylated inall samples, while individual CpG junctions of the genewere equally hypomethylated in the comparison groups.There is evidence of the contribution of miRNA tothe regulation of the RANK/RANKL/OPG system. In2014, C. Chen et al. published results measuring miR-503microRNA levels in peripheral blood cells, the overexpressionof which in CD14+ mononuclear cells inhibitedRANKL-induced osteoclastogenesis. In CD14+ cells frompostmenopausal women with osteoporosis, the baselinelevel of miR-503 was lower than in normal controls andhad no change after induction by RANKL factor, suggestingthe direct role of miR-503 in the regulation ofRANK expression . miR-142-3p andmiR-21- 5p are potential biomarkers of OP as they have ahigh affinity with the OPG gene mRNA and are involvedin the regulation of several signalling pathways involved inbone formation . Thus, theepigenetic regulation of the RANK/RANKL/OPG systemis dynamic and dependent on the DNA methylation statusand a number of microRNAs.Transcription factor 2 (RUNX2) plays a crucial role inosteoblast differentiation. Gene expression is predominantlyhigh in the early stages of bone cell developmentwhen MSCs are differentiating into osteoblast precursors,but naturally decreases at the osteocyte maturation stage.The gene contains several functional regions: activationdomain, runt domain, PST domain, etc. . In terms of epigenetic regulators of RUNX2,microRNAs are well studied. In particular, miRNA-194modulates MSC differentiation  andaccelerates osteoblast differentiation by regulating RUNX2nuclear translocation by way of STAT1 signal transducer. miR-133a-5p inhibits RUNX2 geneexpression at the transcription and translation level by bindingto the 3′-untranslated mRNA site .During the early stages of osteoblast maturation, miR-125b affects RUNX2 expression by affinity binding to the3′-untranslated region of the gene, indirectly participatingin the formation of a complex with Cbfβ that inhibits differentiationof these cells. Using microarray technology,P. Garmilla-Ezquerra et al. (2015) discovered a significantreduction in miR-187-3p gene expression levels andinduction of miR-518f in bone with low BMD. In theirresearch, Y. Zhang et al. (2017) observed the involvementof miR-221 in the formation of low mineral density throughregulation of RUNX2 activity based on bioinformaticsanalysis . Several microRNAs affecting the activity of thisfactor are presented in Table 1.Phosphorylation of RUNX2 mobilises chromatin regulatoryfactors and accelerates MSC maturation. RUNX2 isphosphorylated at specific serine residues 301 and 319, inducingosteocyte maturation through MAPK-dependentsignalling  and BMP2-sensitive transcription . Phosphorylationof S104 leads to prevention of RUNX protein degradation. The ERK-MAPKsignalling pathway plays a crucial role in the regulationof RUNX2 gene expression and bone formation . MKK6 is a protein kinase with dual specificity thatparticipates in the MAP kinase signal transduction pathwayand promotes RUNX2 phosphorylation . Inaddition, parathormone, which is one of the main regulatorsof blood calcium levels, activates the phosphorylation ofthe RUNX2 factor by means of the PKA signalling pathway.This process is associated with the activation of the promoter of the MMP13 gene, which plays an essentialrole in bone resorption .Post-translational modification of histones, in particularhistone methylation, plays an important role in bone formation.The so-called JUMONJI protein is considered to bea transcription factor and is encoded by the JARID2 gene.The domain containing 3 JMJD3 is a histone demethylasespecifically catalysing the removal of trimethylation ofhistone H3K27me3. JMJD3 was found to inhibit the differentiationof RUNX2 osteoblasts. Conversely, inhibitionof JMJD3 activity decreases RUNX2 promoter activitywhile increasing H3K27me3 activity in promoter regions.Histone acetylation affects the state of chromatin compactionby neutralising the positive charge of histone tailsand reducing the electrostatic interactions of histone tailswith deoxyribonucleic acid. Studies have revealed thatosteoporosis induced by glucocorticoid therapy leads todecreased acetylation of H3K9/K14 and H4K12 in theregulatory regions of the RUNX2 and OSX genes andincreases the hyperacetylation of H3K9/K14 and H4K12in the PPARγ2 regulatory region in bone marrow-derivedMSCs from osteoporosis. The transcriptional activity of theRUNX2 factor gene is enhanced by P300 acetyltransferaseand nicotinamide phosphoribosyltransferase (NAMPT),which, in turn, promote osteogenic differentiation of MSCsby H3K14 acylation and MC3T3-E1 cells through H3K9acylation, respectively .Thus, an extremely significant number of regulatorsof RUNX2 gene expression have been identified, whichis a promising therapeutic gene model in culture studies,as blocking or enhancing the activity of this gene andmonitoring its expression level during the induction ofosteogenic lines allows the identification of key switchesof mesenchymal stem cell differentiation.The WNT signalling pathway is one of the most importantsystems regulating embryonic development and cell differentiation.This pathway represents one of the centrallinks in the control of bone development and remodelling.Among the various genes involved in this system, methylationof the SOST (encoding sclerostin) gene promoterhas been comprehensively studied in osteoblast cultures.Sclerostin produced by osteocytes inhibits WNT signallingand reduces the rate of bone formation. DNA methylationof the gene is necessary for the transition of osteoblasts toosteocytes . In women withprimary OP, SOST methylation is elevated in iliac bonecells whilst sclerostin levels are decreased and the WNTpathway is enhanced .Several studies have shown that histone deacetylationunder the regulation of the WNT6, WNT10B, WNT10A andWNT1 genes inhibits WNT signalling and increases the riskof primary osteoporosis . Thus, elevatedlevels of HDAC5 deacetylase reduce SOST gene expressionin osteocytes, contributing to bone mass loss. Deficiency ofthis deacetylase is associated with the acetylation of histoneH3 lysine 27 as well as the interaction of MEF2C with theSOST gene enhancer, therefore suggesting the significantrole of sclerostin in the regulation of osteocyte maturation. High levels of the zeste 2 homologueenhancer methyltransferase (EZH2) have been demonstratedto suppress osteogenic differentiation of MSCs,while low ones decrease the levels of the H3K27me3 tagnear the transcription start site of osteogenesis genes, includingWNT10B . EZH2 increasesH3K27me3 levels at the WNT1, WNT6 and WNT10Apromoters and inhibits WNT signalling .Many regulatory microRNAs are associated with WNTsignalling. miR-433-3p inhibits DKK1 (Dickkopf-1)gene expression, enhancing osteoblast differentiation.Dickkopf-1 acts as an antagonist of the WNT signallingpathway and enhances bone resorption .miR-139-5p induces WNT signalling via the inhibition ofNOTCH1 . R.E. Makitie et al. (2018)screened a specially designed panel of 192 microRNAsin patients with a genetically determined WNT signallingdisorder with a heterozygous missense mutation c. 652 T>G(p. C218G) in exon 4 of the WNT1 gene. It was determinedthat miR-22-3p, miR-34a-5p and miR-31-5p levels werelower in mutation carriers compared to controls .Another common inhibitor of osteogenic differentiationis miR-31, the level of which drops in MSCs differentiatinginto osteoblasts. This was confirmed by S. Weilner et al.(2016), who observed increased levels of this microRNA in plasma in elderly patients with OP . miR-31 isreleased from extracellular vesicles of endothelial cellsand inhibits osteogenesis in stromal stem cells by bindingto the Freisled 3 protein. Furthermore, decreased levels ofmiR-199a-5p result in glucocorticoid-mediated inhibitionof osteogenesis . More recently, L. Duanet al. (2018) identified that high levels of miR-16-2* maycontribute to the development of primary OP: knockdownof this microRNA may promote RUNX2 activation. ThismicroRNA has an affinity for the WNT5A gene mRNA. miR-148a-3p has been revealed toenhance both osteoclastogenesis  andadipogenesis in osteogenic progenitor cells . Plasma levels of this microRNA are significantlyhigher in patients with OP compared to controls withoutOP . miR-30e is another importantmicroRNA in the pathogenesis of OP, playing a role inregulating adipocyte and osteoblast differentiation throughthe inhibition of LRP6 . Thus, the WNTsignalling pathway is regulated by a complex epigeneticsystem, notably microRNAs.The role of non-coding RNAs in bone remodellingMicroRNAs are considered to be the most studied epigeneticfactors in osteoporosis .They are conventionally divided into two classes: those thatpromote bone formation or bone resorption. In particular,several microRNAs that slow down the progression of OPhave been identified. For example, miR-33-5p is a mechanosensitivemicroRNA that positively regulates osteoblastogenesisby way of inhibition of HMGA2 high mobilitygroup proteins . miR-96 enhances osteogenicdifferentiation by inhibiting phosphorylation ofepidermal growth factor receptor EGFR and the expressionof major osteoblast factors RUNX2 and OSTERIX. miR-216a enhances bone formation byregulating the c-Cbl-mediated PI3K/AKT pathway . Table 2 shows the microRNAs separated bydirection of action in bone remodelling .miR-124 is a positive regulator of adipogenic and neurogenicdifferentiation, while being a negative regulator ofmyogenic and osteogenic differentiation. It directly targetsthe DLX3, DLX5 and DLX2 homeobox genes . In one pharmacogenetic study, patients with OP,after three months of treatment with the parathormoneanalogue Teriparatide, had reduced expression levels ofmiR-33 and one year later, miR-133a. Simultaneously,there was a general tendency: the increase in the level ofBMD increased with a decrease in the level of expressionof these microRNAs. Post-transcriptional regulation ofDKK-1 changes due to a decrease in miR-33 microRNAlevels and the action of parathyroid hormone, which leads toa decrease in the negative impact of DKK-1 on an alternativeregulatory mechanism that improves optimal controlof WNT signaling..Of interest are the results of studies on the effects oflong non-coding RNAs on the regulation of Sirtuins,nicotinamide adenine dinucleotide-dependent deacetylases.These molecules have a broad spectrum of action and areassociated with longevity and resistance to age-relateddiseases. It was established that SIRT1 gene expression isinversely related to HIF1A-AS1 ncRNA expression, whileHOXA-AS3 ncRNA interacts with EZH2 and is requiredfor RUNX2 trimethylation of lysine-27 H3 (H3K27me3)factor. Hence, HOXA-AS3 is important for bone formationin general .The long ncRNA HOTAIR reduces protein expressionand inhibits WNT signalling. DKK1 reduces the proteinlevels of C-myc, β-catenin, HOTAIR and RUNX2, whichtheoretically counteracts the regulatory effect of HOTAIR. If the expression level of p21 ncRNA islow, WNT signalling becomes more active due to increasedE2 secretion, which ultimately increases the rate of boneformation . Also, reduced levels of H19ncRNA reduce the level of DKK4 gene expression . AK045490 ncRNA levels are significantlyelevated and inhibit bone formation by inhibiting nucleartranslocation of β-catenin and suppressing TCF1, LEF1 andRUNX2 expression . Similarly, AK016739ncRNA inhibits osteogenic differentiation as it can reducethe expression and activity of osteoblastogenesis transcriptionfactors .Inhibition of UCA1 ncRNA promotes bone formationvia activation of the BMP-2/(Smad1/5/8) pathway in osteoblasts. As a result, microRNAs andlong ncRNAs remain among the most studied epigeneticfactors involved in the pathogenesis of primary OP butrequire further systematisation.The mechanisms of the relationship between bone andadipose cell formation are complex and remain an area ofactive research. Works performed on the cell cultures ofosteoblasts and MSCs convincingly show an inverse relationshipbetween differentiation of bone marrow MSCs intoadipocytes or osteoblasts. An imbalance between adipogenesisand osteogenesis has been proposed as a mechanismfor the development of OP, but obesity itself is not alwaysa predictor of an increased risk of osteoporosis.Post-translational modifications of histones play a keyrole in this system. Among them, histone methylation iscrucial, in particular in chromatin reorganisation. In particular,lysine methylation in H4K20, H3K27 and H3K9 isassociated with decreased levels of transcription, whereasmethylation of H3K79, H3K36 and H3K4, with active genetranscription .However, concerning osteogenic inducers, the homeoboxgene HOXA10, which contributes to osteogenic clone determinationby enriching the trimethylation of the 4th lysineresidue in histone, activating RUNX2, alkaline phosphataseand osteocalcin, thus stimulating bone cell maturation, isimportant . It is known that the combinationof methylation and demethylation can function as anepigenetic switch of osteogenesis to adipogenesis based onEZH2 activity, catalysing the trimethylation of histoneH3on lysine 27 key regulatory genes (such as RUNX2). Simultaneously,removal of this tag by lysindemethylase 6Ainhibits adipogenesis and induces osteoblastogenesis. Proteinsthat can be targeted by EZH2 and are involved in MSCswitching are HDAC9c and HDAC .A direct correlation was realised between increased levelsof EZH2 and decreased levels of HDAC9c gene expression. The methyltransferase activity ofEZH2 is reduced by phosphorylation and is associated withosteogenic induction It is acknowledged that during osteocyte aging there isan accumulation of adipose tissue in the bone marrow and,at the same time, the number of mesenchymal stem cellsincreases in the intercellular phase. From this perspective,it is interesting that overexpression of miR-1292 acceleratesthe senescence of human adipose-derived stem cellsand inhibits bone formation via the Wnt/β-catenin signallingpathway, while miR-10b inhibits adipose stem celldifferentiation via the TGF-β pathway .Several bone-associated cells, including multipotentbone mesenchymal stem cells, osteoblasts that form bonetissue and osteoclasts that break it down, are in a symbioticrelationship throughout life. A growing body of evidencesuggests that epigenetic cell modifications induced byaging contribute to impaired bone remodelling and leadto osteoporosis. A variety of epigenetic mechanisms areinvolved, including DNA/RNA modifications, histonemodifications, microRNAs (miRNAs) and long non-codingRNAs (dnRNAs), and chromatin remodelling . Thus, epigenetic mechanisms can switch the directionof mesenchymal stem cell differentiation betweenosteoblastogenesis and adipogenesis.Molecular genetic determinants of endophenotypes ofosteoporosis, such as fracture risk and BMD levels, can beconverged through the whole epigenome. In the researchthey conducted, J.A. Morris et al. (2017), leveraging thetechnological capabilities of Infinium HumanMethylation450,performed a whole-genome methylome analysis,measuring site-specific DNA methylation in 5515 individualsof European descent. Following a meta-analysisof their results, they were able to identify the CpG sitecg23196985, which was significantly associated with lowMPCT adjusted for multiple comparisons without regard togender( pBH = 1.30 × 10–2) and in females (pBH = 3.41 × 10–5).The CpG cg23196985 site is localized to the 5′-translatedregion of the hepatic carboxylase 1 gene CES1, which isexpressed in the liver and peripheral blood . J.G. Zhang et al. (2015), performing transcriptomeanalysis based on Affymetrix GeneChip Human Exon 1.0ST Array and microRNA analysis on Capitalbio Cor. microarrays,as well as methylome sequencing in patientswith low MPCT hip and controls, identified the mostenriched functional molecular pathways associated withOP or MPCT variability: a network of 12 interacting genesand 11 microRNAs. Among the genes are AKT1, STAT5A,PIK3R5, etc., while among the microRNAs, miR-141 andmiR-675 – their levels correlate with the expression ofthese genes and global DNA methylation status .D. Cheishvili et al. (2018) performed a full-epigenomicanalysis in women without OP and in women with earlypostmenopausal OP. Genes in which CpG sites with significantlevels of differential methylation were identifiedwere ZNF267, ABLIM2, RHOJ, CDKL5, PDCD1, ABRAand HOJ . At the human femurlevel, DNA methylation profile studies using pyrosequencingand qRT-PCR-based gene expression studies provedthat DNA methylation status was inversely correlated withthe expression of the iNOS and COL9A1 genes, but notcatabolic genes including MMP13 and IL1B. Significantdemethylation of the osteocalcin gene promoter was alsoshown between the embryonic and adult stages of development,demonstrating the importance of DNA methylation atthe tissue level . It is anticipated that theresults of these studies will confirm the whole-epigenomeapproach as being sufficiently robust to allow large-scalestudies of women at risk of developing OP.Despite the great advances and the wide range of workperformed in the study of the epigenetics of primary osteoporosis, understanding remains fragmentary. The researchdescribes key epigenetic regulators of bone remodelling,but it is difficult to build a coherent picture of the pathogenesisof osteoporosis from these data, common to all keypathogenetic processes in bone tissue.It is recognised that the key epigenetic changes in osteoporosisare converging on the regulation of MSCs differentiationand that the multi-step system of activity regulationof the transcription factor RUNX2, sclerostin,DKK1 protein, RANKL-RANK-OPG system and factorsinvolved in the regulation of WNT signalling is of greatimportance. A separate issue is the systematisation andcreation of a unified genetic network of a vast number ofregulatory microRNAs that affect key signalling pathwaysand the transcription factors associated with osteoporosis.However, these microRNAs influence the ultimate risk ofosteoporosis indirectly and it remains to be understoodhow they can be systematised for relevance and functionalinvolvement in pathogenesis due to their dynamism anddifferent levels depending on tissue specificity.Approaches necessary to implement early diagnosis ortargeted therapies for osteoporosis still need to be developed.The task is complicated by experimental data frommethylome studies, in which genes other than critical regulatoryfactors, such as RUNX2, sclerostin or DKK1, wereobserved to be key and most important. 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Enterobacterales (MDR-Ent) has reached worrisome levels worldwide. In this context, Escherichia ruysiae is a recently described species mostly found in animals. However, its spread and impact on humans is poorly understood. A stool sample from a healthy individual living in India was screened for the presence of MDR-Ent using culture-based methods. Colonies were routinely identified using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and phenotypically characterized by broth microdilution. Illumina and Nanopore whole-genome sequencing (WGS) platforms were implemented to generate a complete assembly. E. ruysiae genomes deposited in international databases were used for a core genome phylogenetic analysis. An extended-spectrum β-lactamase (ESBL)-producing E. coli strain (S1-IND-07-A) was isolated from the stool. WGS confirmed that S1-IND-07-A was indeed E. ruysiae, belonged to sequence type 5792 (ST5792), core genome (cg) ST89059, serotype O13/O129−:H56-like, clade IV phylogroup, and possessed five virulence factors. A copy of blaCTX-M-15 and five other antimicrobial resistance genes (ARGs) were detected in a conjugative IncB/O/K/Z plasmid. A database search identified 70 further E. ruysiae strains from 16 countries . The core genome phylogeny revealed five major STs: ST6467, ST8084, ST2371, ST9287, and ST5792. Three out of the seventy strains possessed important ARGs: OTP1704 , SN1013-18 , and CE1758 . These strains were of human, environmental, and wild animal origin, respectively. E. ruysiae may acquire clinically important ARGs and transmit them to other species. Due to its zoonotic potential, further efforts are needed to improve routine detection and surveillance across One Health settings.Gut colonization with multidrug-resistant IMPORTANCEEscherichia ruysiae is a recently described species of the cryptic clades III and IV of the genus Escherichia and is commonly found in animals and the environment. This work highlights the zoonotic potential of E. ruysiae, as it has been shown to colonize the human intestinal tract. Importantly, E. ruysiae may be associated with conjugative plasmids carrying clinically relevant antibiotic resistance genes. Therefore, it is important to closely monitor this species. Overall, this study highlights the need for improved identification of Escherichia species and continued surveillance of zoonotic pathogens in One Health settings. Escherichia is comprised of 3 monophyletic species , including cryptic clades I, II, III and IV (E. ruysiae), and V (E. marmotae)  corresponding to specific host/source niches where this pathogen can be found, such as commensal E. coli of the gastrointestinal tract in humans (groups A or B1) and animals (group B1) and the environment  , such as those encoding extended-spectrum β-lactamases , which compromise the treatment (The genus armotae) –3. FurthB2 or D) . On thisreatment .E. ruysiae has been recently reported from animal and environmental sources, but it is still very rarely isolated in humans , unless a PCR-based approach  or whole-genome sequencing (WGS) are used  blaCTX-M-14-possessing Escherichia cryptic clade IV strain (OPT1704) was recently isolated from the stool of a traveler to Asia  isolated from a fecal sample of a healthy individual living in India. To investigate the spread of E. ruysiae in humans, we searched for other E. ruysiae genomes in international databases and compared the results using core genome phylogenetic analysis. Furthermore, we investigated the distribution of the associated ARGs, plasmids, virulence factors, and serotypes characteristic of strains isolated from various human and nonhuman samples.In this work, we characterized a E. ruysiae strain isolated from a person living abroad. To contextualize our findings, the second aim was to search global databases for other reported E. ruysiae strains isolated from different sources to perform a core genome phylogeny analysis. Finally, we examined the associated ARGs, plasmid replicons, virulence, and serotype distribution of all E. ruysiae strains included in this study to evaluate the potential of E. ruysiae as a human pathogen.The first aim of this study was to characterize a rare ESBL-producing E. coli . As shown in Screening of the individual’s stool yielded a third-generation cephalosporin-resistant strain (S1-IND-07-A) that was identified by MALDI-TOF MS as E. ruysiae . This was also supported by in silico phylotyping, which determined that the species belonged to Escherichia cryptic clade IV.Using WGS, the species was confirmed as blaCTX-M-15, aadA1, dfrA1-like (99.79% identity), qnrS1, sul2, and tet(A). In the chromosome, mutations encoding for the quinolone resistance-determining region substitution (S83A) in GyrA were identified, together with the mdf(A)-like ARG (91.32% identity) . Overall, the genotypic screening results were consistent with the observed phenotype (S1-IND-07-A was of sequence type 5792 (ST5792) and core genome (cg) ST89059. Its genome consisted of a 4.4-Mb chromosome and 2 circular plasmids of 105.8 kb (IncB/O/K/Z) and 1.5 kb [Col(MG828)]. Most ARGs were carried by the IncB/O/K/Z plasmid (p1-S1-IND-07-A): henotype .in silico serotyping revealed that S1-IND-07-A carried O13/O129-like  and H56-like  antigens. Several virulence factor genes were also identified, including those encoding colonization factors (fimH), fitness (iutA and kpsMII), and toxins (pic and sat) (Table S1). Of note, p1-S1-IND-07-A carried the colicin Ib virulence factor gene cib, which is known to enhance virulence  in, for example, E. coli and Salmonella enterica (Table S1)  –13.E. ruysiae strains of the same sequence type and core genome sequence type (ST5792 and cgST89059). We also included strains isolated from different sources to identify potential ARG hot spots and evaluate the pathogenic potential of E. ruysiae in humans. Overall, we included a total of 70 E. ruysiae genomes with complete metadata  from the NCBI  and Enterobase  databases.To investigate the origin of S1-IND-07-A, we performed a database search for other E. ruysiae strains belonging to the cryptic clades III (n = 36) and IV (n = 35), detected between 1989 and 2022 , environmental , and human sources . The strains originated from 16 countries, mainly from the United States (n = 31) and the United Kingdom (n = 16).As a result, along with our strain, we identified a total of 71 n = 9), ST8084 (n = 8), ST2371 (n = 6), ST9287 (n = 4), and ST5792 (n = 4)  were observed. Overall, they consisted of strains isolated from poultry in the UK (C17-1 and C10-7), companion animals in Australia (MVC117 and MVC118), and the environment in the United States  –17.–E. ruysiae strains from clade IV carried β-lactamase (bla) genes: CE1758 (blaCMY-2), OPT1704 (blaCTX-M-14 and blaLAP-2), and SN1013-18 (blaCTX-M-15 and blaTEM-1). The blaCTX-M-14 and blaLAP-2 genes in OPT1704 were associated with an IncI plasmid, while the blaCMY-2, blaCTX-M-15, and blaTEM-1 genes in CE1758 and SN1013-18 were unspecified (As depicted in mdf(A) and  sitABCD. Furthermore, many replicon sequence types were detected in both clades III and IV, most frequently Col and IncFI type  or O36-like (n = 10) and H42-like (n = 15), H52-like (n = 11) and H56-like (n = 10), while clade IV strains were O36-like (n = 11) or O13/O129-like (n = 8) and H56-like (n = 20). Furthermore, many virulence factors were also detected, but only a few were clinically relevant, as they were related to host colonization (fimH), fitness (iutA and kpsMII), toxins , and effectors (aaiC)  and pic and sat . Both estB and astA genes have been reported in enterotoxigenic E. coli strains in both diseased pigs and pigs with diarrhea, while pic and sat have been found in enteroinvasive E. coli strains . Compared to p1-S1-IND-07-A, the other plasmids were missing an IS26 flanking region containing tet(A) and sul2 and including a disrupted class I integron (missing sul1) region associated with the dfrA1-aadA1-qacEΔ1-IS26-ΔblaTEM-1 cassette. Similar integron regions have been reported in hyperepidemic carbapenemase-producing E. coli strains also carrying blaTEM-30-associated IncI1 plasmids from colonized employees of a Swiss veterinary clinic (In our is study . Howevery clinic .blaCTX-M) and multiple hosts   –27. In f J53d-R1 , thus coEnt in the gut, which may include zoonotic diseases that can be transmitted between humans, animals, and the environment  and transfer them to other clinically important bacteria. Therefore, the application of WGS surveillance methods  is essential for the discovery and characterization of novel Escherichia species with zoonotic potential and the ability to serve as reservoirs for resistance plasmids. In this context, it is crucial to develop and improve routine laboratory identification techniques such as MALDI-TOF MS to distinguish between Escherichia species.In the present work, we showed that Enterobacterales (MDR-Ent) on his return to Switzerland in 2022 as part of an ongoing prospective screening study (https://data.snf.ch/grants/grant/192514). The stool sample was collected by the volunteer in a sterile container and sent within 1 day to the Institute for Infectious Diseases in Bern for processing (see below). No hospitalization or diarrhea was reported, but previous antibiotic use (not specified) was noted in two independent episodes 2 and 4 months before arrival in Switzerland. The study participant provided written consent, and the study was approved by the ethical committee of the Canton Bern, Switzerland (2020-01683).A healthy 62-year-old Swiss man living in India for 1.3 years was screened for intestinal carriage of multidrug-resistant A stool sample (~50 μg) was preenriched for 6 h in 10 mL Luria-Bertani (LB) broth supplemented with cefuroxime . The preenrichment was then diluted to a 0.5 McFarland standard, and a 100-μL aliquot was plated overnight at 36 ± 1°C on a CHROMID ESBL agar plate (bioMérieux). The resulting colonies were subcultured on MacConkey II agar  overnight at 36 ± 1°C for further processing.Enterobacterales .Species identification of overnight-grown colonies was conducted using MALDI-TOF MS . Antimicrobial susceptibility testing (AST) was performed by broth microdilution implementing the Sensititre GNX2F panel (Thermo Fisher Scientific). Results were interpreted according to the 2023 European Committee on Antimicrobial Susceptibility Testing (EUCAST) v13.0 breakpoints for https://github.com/usadellab/Trimmomatic) and Porechop v0.2.4 (https://github.com/rrwick/Porechop), respectively. Long reads were filtered by read quality using Filtlong v0.2.1 (https://github.com/rrwick/Filtlong) . A complete genome assembly was generated using the hybrid pipeline from Unicycler v0.4.8 (https://github.com/rrwick/Unicycler), and its completeness  was confirmed with an independent long-read-only assembly using Flye v2.9-b1768 (https://github.com/fenderglass/Flye) (parameters: “–nano-raw” and “–iterations 5”). Genome assembly coverage was calculated using QualiMap v2.2.2-dev .Genomic DNA for WGS was extracted from strain S1-IND-07-A using the Invitrogen PureLink microbiome DNA purification kit (Thermo Fisher Scientific), and the quality was assessed using NanoDrop and Qubit 3 (Thermo Fisher Scientific). Short-read WGS was performed on a NovaSeq 6000 platform , while long-read WGS was conducted on a MinION sequencer (Oxford Nanopore) using a rapid barcoding library prep (SQK-RBK004) and a FLO-MIN 106D R9 flow cell, as described previously –31. In b–E. coli number 1 configuration), cgMLSTFinder v1.2 (parameter: E. coli [Enterobase]), SerotypeFinder v2.0, and VirulenceFinder v2.0 (parameters: 60% minimum coverage and 95% minimum identity), respectively, from the Center for Genomic Epidemiology (https://www.genomicepidemiology.org/). In addition, the phylogroup was determined using the ClermonTyping tool (Clermont Type) available at Enterobase (https://enterobase.warwick.ac.uk/) (Escherichia/Shigella database). Finally, the species ID was confirmed using the Type (Strain) Genome Server (TYGS) (https://tygs.dsmz.de/) and by ANIb (based on BLAST) using JSpeciesWS (https://jspecies.ribohost.com/jspeciesws/) with the genome assembly.The genome assembly of S1-IND-07-A was screened for ARGs, plasmid replicon sequences, sequence type (ST), core genome ST (cgST), serotype, and virulence using ResFinder v4.1, PlasmidFinder v2.1 (parameters: 60% minimum coverage and 50% minimum identity), MLST v2.0 (parameter: https://isfinder.biotoul.fr/) with the BLASTn algorithm. Circular BLASTn plasmid comparisons were performed using BLAST Ring Image Generator v0.95 (https://brig.sourceforge.net/) with the most similar plasmids retrieved from NCBI BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and PLSDB v2021_06_23_v2 (https://ccb-microbe.cs.uni-saarland.de/plsdb/) on 27 January 2023.The genome assemblies were automatically annotated using the NCBI Prokaryotic Genome Annotation Pipeline. Insertion sequences (ISs) were manually confirmed using ISfinder  and Enterobase (https://enterobase.warwick.ac.uk/species/index/ecoli)  databases on 30 January and 2 March 2023, respectively. The species IDs of all the retrieved genomes were confirmed using the TYGS tool as described above.GCA_902498915.1) as the mapping reference. Genome regions with recombination were detected and removed using Gubbins v2.3.4 (https://github.com/nickjcroucher/gubbins). Furthermore, IS regions were identified with ISEScan v1.7.2.3 (https://github.com/xiezhq/ISEScan) and subsequently used to mask single nucleotide variants (SNVs) localized within these regions . A maximum likelihood core genome phylogeny (unrooted) was inferred using IQ-TREE v2.1.2 (http://www.iqtree.org/) with the general time reversible (GTR) model with ascertainment bias correction (parameters: GTR + ASC), 1,000 ultrafast bootstraps , and the SH-aLRT test . The tree was visualized and annotated using iTOL v6.7.2 (https://itol.embl.de/). Unless otherwise noted, all software analyses described above were performed with default parameters.A core genome alignment was performed using Snippy v4.4.5 with strain OPT1704 (GenBank assembly accession number E. coli recipient strain J53d-R1 was conjugated with the donor S1-IND-07-A as previously described (blaCTX-M group I primers (The rifampicin-resistant escribed . In shor primers . ExperimCP112983 to CP112985) and under BioProject accession number PRJNA903117.The complete genome sequence of S1-IND-07-A has been deposited at GenBank (accession numbers


Healthcare personnel (HCP) are at risk for acquiring and transmitting coronavirus disease 2019 (COVID-19) at work. Vaccination against COVID-19 is a primary strategy to prevent transmission in healthcare settings. Our objectives were to describe HCP reporting employer COVID-19 vaccine requirements, identify differences across occupations and settings, and describe policies.In a national opt-in internet panel survey of HCP in April 2022 to assess influenza and COVID-19 vaccination coverage, respondents were also asked about employer vaccination requirements. We weighted responses to the U.S. HCP population by age, sex, race, ethnicity, work setting, and U.S. census region. We assessed differences using two-tailed t tests with significance set at p< 0.05. We used multivariable logistic regression to identify factors associated with reporting employer requirements.Of 3,618 responding HCP, 60.0% reported employer requirements for the COVID-19 primary series. Of those, 95.5% reported completing the primary series vs. 81.9% without requirements (p< 0.001). In addition, 24.0% of HCP reported employer requirements for a booster dose. Of those eligible, 90.8% reported receiving a booster vs. 60.2% without a requirement (p< 0.001). Employer requirements for the primary series varied by occupation [46.1% (assistants/aides) to 77.5% (nurses)] and work setting [40.4%  to 75.4% ]. Of 2,157 HCP reporting a primary series requirement, employer accepted reasons for not being vaccinated included medical (56.8%), religious (50.9%), philosophical (10.0%), all three reasons (16.7%), and no reason (14.9%). Factors independently associated with no reported primary series requirements included working as an assistant/aide, in a non-clinical or other clinical occupation, in ambulatory care, long-term care/home health, or other clinical setting, and in the Midwest and South.Most HCP in this sample reported employer requirements for the COVID-19 vaccination primary series although requirements were less common for boosters. Differences by healthcare setting exist. Implementing workplace strategies, including vaccine requirements, may lead to higher COVID-19 vaccination coverage.All Authors: No reported disclosures


Wikstroemia indica, a plant belonging to the Thymelaeaceae family, was investigated by LC-MS/MS analysis. As a result, 21 daphnane diterpenoids (1–21) in the stems of W. indica were detected. Among these, six major compounds  were isolated and their structures were unequivocally identified through a comprehensive analysis of the MS and NMR data. For the minor compounds , their structures were elucidated by in-depth MS/MS fragmentation analysis. This study represents the first disclosure of structurally diverse daphnane diterpenoids in W. indica, significantly contributing to our understanding of bioactive diterpenoids in plants within the Thymelaeaceae family.Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for the rapid identification of compounds within natural resources. Daphnane diterpenoids, a class of natural compounds predominantly found in plants belonging to the Thymelaeaceae and Euphorbiaceae families, have attracted much attention due to their remarkable anticancer and anti-HIV activities. In the present study, the presence of daphnane diterpenoids in  Liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HR-MS/MS), usually equipped with an electrospray ionization source, has high adaptability across a broad spectrum of compounds, offering high mass accuracy and sensitivity. Moreover, it provides information-rich fragmentation through product ion spectra, thereby potentially revealing details about the molecular formula and structure of diverse secondary metabolites found in plants . The contrans-fused 5/7/6-tricyclic skeleton, have garnered attention for their diverse biological activities, including anticancer +) were observed in positive ion mode, while deprotonated molecular ions ([M–H]−) and/or formate adduct ions ([M + HCOO]−) were observed in negative ion mode. (2) In the product ion spectrum obtained from the protonated molecular ion as a precursor ion, a diagnostic ion at m/z 253 (C17H17O2) or 269 (C17H17O3) was observed in the positive ion mode . Th. Th12, 1e G (12) , wikstro + appeared with greater intensity in the mass spectrum of peak 3  + (1), m/z 1028.4264 [M + NH4]+ (2), m/z 585 [M + H–H2O]+ (3), m/z 547.2888 [M + H]+ (4), m/z 655.3469 [M + H]+ (5), m/z 719.3062 [M + H]+ (6), m/z 533.3104 [M + H]+ (7), m/z 775.3690 [M + H]+ (8), m/z 691.3133 [M + H]+ (9), m/z 721.3203 [M + H]+ (10), m/z 775.3687 [M + H]+ (11), m/z 912.4147 [M + NH4]+ (12), m/z 667.3107 [M + H]+ (13), m/z 673.2999 [M + H]+ (14), m/z 533.3107 [M + H]+ (15), m/z 557.3109 [M + H]+ (16), m/z 675.3151 [M + H]+ (17), m/z 639.3527 [M + H]+ (18), m/z 643.3458 [M + H]+ (19), m/z 639.3525 [M + H]+ (20), and m/z 585.3409 [M + H]+ (21). All data collected in the profile mode were acquired and processed using Thermo Xcalibur 4.1 software.The LC-MS/MS analysis was performed using the same instruments and column as in previous experiments . For LC W. indica through a combination of LC-MS guided isolation and MS/MS fragmentation elucidation. The investigation revealed that W. indica contained structurally diverse daphnane diterpenoids, including orthoester daphnane type, polyhydroxy daphnane type, and macrocyclic daphnane orthoester type compounds. The application of MS/MS fragmentation elucidation for structural analysis enabled the rapid and precise identification of these diterpenoids within crude plant extracts. This methodology holds great promise for future research endeavors aimed at discovering bioactive diterpenoids from plants of the Thymelaeaceae family.This study represents the first comprehensive identification of 21 daphnane diterpenoids from the stems of


Correction: Respiratory Research (2022) 23:291 10.1186/s12931-022-02195-3Following the publication of the original article , it was The original article has been corrected.Additional file 8: Table S1. The RECORD statement—checklist of items, extended from the STROBE statement, that should be reported in observational studies using routinely collected health data.Additional file 9: Table S2. Disease names adopted as inclusion criteria.Additional file 10: Table S3. Disease names adopted as exclusion criteria.


Mycobacterium marinum strain, which was isolated from skin tissue of a wound infection. This strain was subjected to short- and long-read sequencing. Its complete genome contains a single 6,393,703-bp circular chromosome. Phylogenomic analysis of all M. marinum genomes assigned this strain to cluster I.We report the complete genome sequence of a  Mycobacterium marinum is an opportunistic human pathogen  were filtered using NanoFilt v2.8.0 (de novo using Canu v2.2 (https://github.com/nanoporetech/medaka).Genomic DNA was extracted using a QIAamp minikit. Large DNA fragments were recovered using a Blue Pippin recovery system. Barcodes were added using PCR-free EXP-NBD104, adapters were ligated using a SQK-LSK109 connection kit (Oxford Nanopore), and long-read sequencing was performed using the Nanopore PromethION platform. Raw Nanopore signals were subjected to base calling, demultiplexing, and adapter trimming using Guppy v6.3.9  with a sanu v2.2 , Flye v2https://github.com/usadellab/Trimmomatic) and were used to polish the Nanopore assembly with two alternating rounds of Polypolish v0.5.0 . Paired reads  were trimmed using Trimmomatic v0.39 , M. pseudoshottsii JCM15466T (AP018410.1), M. shottsii JCM12657T (AP022572.1), and M. ulcerans Agy99T (CP000325.1) was 98.56%, 98.36%, 97.70%, and 98.09%, respectively, as determined using FastANI v1.33  rotated to start with NI v1.33 . Neverthm 050012 , 18, conM. marinum genome assemblies (n = 51) and SRA reads (n = 88) from NCBI. SRA reads were trimmed using Trimmomatic and assembled de novo using SPAdes v3.15.3 (n = 26) were removed. All genomes (n = 113) were identified as M. marinum as described above. Single nucleotide polymorphisms (SNPs) were called using M. marinum CCUG20998T as a reference using Snippy v4.6.0 (https://github.com/tseemann/snippy) with --ctgs option and Gubbins v3.2.1 with a general time-reversible (GTR) model plus gamma distribution and a 100-bootstrap test (M. marinum strains belong to two major clusters (I and II), consistent with previous findings  and SRR23294010 (Illumina reads), and GenBank nucleotide accession number CP118910.Complete genome sequences of


Scientific Reports 10.1038/s41598-023-33920-7, published online 27 April 2023Correction to: The original version of this Article contained errors in the Materials and methods section, under the subheading ‘Software and hardware specification’, where links to used resources were omitted and the present link was incorrect.https://docs.nvidia.com/deeplearning/frameworks/tensorflow-release-notes/rel_21-03.html).”“The sMRI preprocessing was done through the SPM12 v7771 software. All further steps used Python 3.8.5 and Tensorflow 2.4.0. The machine learning experiments were performed on an NVIDIA DGX-2 server, within a Docker virtual machine containing 4 CPUs @2.7Ghz and 1 GPU TESLA V100-SXM3-32 GB. All source codes are available at Github (now reads:https://www.fil.ion.ucl.ac.uk/spm/software/spm12/). All further steps used Python 3.8.5 and Tensorflow 2.4.0 (https://docs.nvidia.com/deeplearning/frameworks/tensorflow-release-notes/rel_21-03.html). The machine learning experiments were performed on an NVIDIA DGX-2 server, within a Docker virtual machine containing 4 CPUs @2.7Ghz and 1 GPU TESLA V100-SXM3-32 GB. All source codes are available at Github .”“The sMRI preprocessing was done through the SPM12 v7771 software (The original Article has been corrected.


Adherence to antiretroviral therapy (ART) is critical for persons with HIV (PWH) for achieving durable virologic suppression and minimizing drug resistance. This study aimed to characterize ART adherence and its effect on quality of life (QoL) among PWH using real-world data.Data were drawn from the Adelphi HIV Disease Specific Programme™, a cross-sectional survey of physicians and PWH in the U.S., collected between July-2021 and March-2022. At consultation, each of 60 physicians reported demographics, clinical characteristics, ART adherence and satisfaction on 10 consenting PWH. These PWH were then invited to complete questionnaires reporting QoL . Physicians categorized adherence as completely adherent (C-Adh), mostly adherent (M-Adh), or less adherent (L-Adh). Comparisons were made using Chi squared test.Of 578 PWH with physician-reported adherence status; mean [SD] age 44.9 [12.8] years, 70.4% cisgender male, 50.5% White. A total of 389 (67%) were C-Adh, 155 (27%) M-Adh, and 34 (6%) L-Adh. L-Adh (55.9%) and M-Adh (46.5%) PWH had the higher proportion of ART side effects compared to C-Adh (29.3%); p< 0.001. Also, L-Adh (75.8%) and M-Adh (50.8%) were more likely to report current HIV-associated symptoms than C-Adh PWH (29.3%); p< 0.001. C-Adh PWH were perceived as very satisfied with their ART (68.4%), compared to M-Adh 39.4% and L-Adh 23.5%; p< 0.001. Reasons for PWH skipping ART included forgetting, difficulties integrating into routine, and ART side effects . QOL survey was completed by 229 PWH. QoL was higher for C-Adh PWH, measured by PozQol score mean,  and EQ-5D score mean .About one-third of PWH who were not fully adherent to ART, and this was correlated with having poorer QoL. These findings highlight the need to better understand factors leading to sub-optimal ART adherence, to provide targeted interventions to improve quality of life.Girish Prajapati, M.B.B.S., MPH , Merck & Co., Inc.: Employee|Merck & Co., Inc.: Stocks/Bonds Bekana K. Tadese, PhD, MPH, Merck and Co Inc: Employee


We report the genome sequences of two genetic variants of grapevine rupestris stem pitting-associated virus (GRSPaV) from Idaho, USA. The coding-complete, positive-strand RNA genome of 8,700 nucleotides contains six open reading frames characteristic of foveaviruses. The two Idaho genetic variants belong to GRSPaV phylogroup 1. Foveavirus (family Betaflexiviridae) (Vitis vinifera L. and its hybrids (––Grapevine rupestris stem pitting-associated virus (GRSPaV) has flexuous filamentous particles and belongs to the genus viridae) . GRSPaV viridae) . The vir hybrids –4. The vhybrids –, 5, 6; ihybrids ––9.V. vinifera reference genome using bowtie2 v2.4.4 in local mode  from British Columbia, Canada. The same Riesling sample also yielded three additional contigs of 2,514, 2,893, and 506 nt, which exhibited 98.3%, 98.6%, and 98.8% identity, respectively, to the GRSPaV isolate AMCF clone 3 (GenBank accession number MG938311) from France  contig (GRSPaV-ID1) that spanned six ORFs and, based on the BLASTn program , exhibited 98.5% identity to the GRSPaV isolate 12G412  under BioProject accession number PRJNA931750.The coding-complete genome sequences of GSPaV-ID1 and GSPaV-ID2 are available in GenBank under accession numbers


Invasive fungal infections present a significant risk to human health. The current arsenal of antifungal drugs is hindered by drug resistance, limited antifungal range, inadequate safety profiles, and low oral bioavailability. Consequently, there is an urgent imperative to develop novel antifungal medications for clinical application. This comprehensive review provides a summary of the antifungal properties and mechanisms exhibited by natural polyketides, encompassing macrolide polyethers, polyether polyketides, xanthone polyketides, linear polyketides, hybrid polyketide non-ribosomal peptides, and pyridine derivatives. Investigating natural polyketide compounds and their derivatives has demonstrated their remarkable efficacy and promising clinical application as antifungal agents. Candida, Cryptococcus, and Aspergillus species . Th. ThFusar B1 (87) . The C. toxicity . Parnafud D (93) , analogstructure . Parnafu 5 μg/mL  (Table 194)  shows antifungal activity against C. albicans, C. neoformans, and S. cerevisiae, with MIC values of 2, 2, and 15.6 μg/mL, respectively [94) has been shown to possess fungicidal activity against C. albicans, C. neoformans, and S. cerevisiae with minimum fungicidal concentrations of 4, 4, and 15.6 μg/mL, respectively [95) (S. cerevisiae (MIC > 200 µM), indicating the importance of the aldonic acid group for the antifungal activity of khafrefungin (94) [96) (94), indicating that the aldonic acid group not only enhances the water solubility of khafrefungin (94) but also plays a role in its antifungal activity [97) (94), highlighting the essentiality of the configuration of the 4-methyl group for the antifungal activity of khafrefungin (94) [94) under acidic conditions forms a six-membered lactone derivative (98) (S. cerevisiae (MIC = ~10 µM) to that of native khafrefungin (94) [The polyketide chain skeleton was catalyzed by type I PKSs and then subjected to complex post-modifications, including glycosylation, to form linear polyketide . There a94) , isolateaffected . Khafrefectively . Additioectively . Notablyely [95)  greatly gin (94) . The pre94) [96)  has beenactivity . Furtherity [97)  has beengin (94) . Additioive (98)  that exhgin (94)  Table 194 (S. ceBacillus laterosporus PNG 276 [99) (62) were administered to human diploid fibroblasts, with basiliskamide A (99) exhibiting minimal cytotoxicity at a concentration of 100 μg/mL, while cytopathic effect was observed with amphotericin B (62) only 12.5 μg/mL [99) demonstrated antifungal activity against C. albicans and A. fumigatus, with MIC values of 1 and 2.5 μg/mL, respectively, whereas basiliskamide B (100) (101) (99), inhibits the growth of C. albicans at a concentration of 25 μg/mL and has no effect on the growth of A. fumigatus at a concentration of 50 μg/mL [101) exhibits an additional methylene group compared to the linear polyketide chain of basiliskamide A (99), potentially accounting for the diminished antifungal activity of YM-45722 (101) [Basiliskamide A and B are isolated from  PNG 276 . Basilis276 [99)  and amph.5 μg/mL . Further B (100)  exhibite0) (101) , an anal50 μg/mL  (Table 122 (101) .FKS1 and FKS2 genes results in the inhibition of biosynthesis of β--D-glucan, an important component of the fungal cell wall, which destroys the integrity of fungal cell wall and disrupts the osmotic balance, ultimately leading to fungal death [Candida and Aspergillus species, but they are ineffective against C. neoformans [0 (102) (103) (0 (102) are discussed in this section. Pneumocandin B0 (102) is isolated from the filamentous fungus Glarea lozoyensis [0 (102) is a lipopeptide composed of myristic acid and a hexapeptide ring. PKSs catalyze the assembly of 10, 12-dimethylmyristic acid, and then, catalyzed by a series of enzymes, the polyketide intermediate localizes to NRPSs to acylate the 4, 5-dihydroxyornithine of pneumocandin B0 (102), initiating cyclic hexapeptide elongation.Echinocandins are novel lipopeptide antifungal products synthesized by a heterozygous pathway of non-ribosomal peptides synthases (NRPSs)-PKSs. Compared with azole and polyene antibiotics, echinocandins have a completely different mechanism of action and exert their antifungal effect by destroying the cell wall. Non-competitive binding of echinocandins to the catalytic subunits of β--D-glucan synthetase encoded by the al death ,83. Echioformans   , the lea2) (103) , is assezoyensis ,88. Pneu104) and 1097 (105) (Burkholderia ambifaria 2.2N [S. cerevisiae (MIC = 0.4 μg/mL), C. albicans (MIC = 12.5 μg/mL), and A. niger (MIC = 12.5 μg/mL) [S. cerevisiae (MIC = 1.6 μg/mL), A. niger (MIC = 1.6 μg/mL), and C. albicans (MIC = 12.5 μg/mL). Burkholdine 1215 (106), 1119 (107), and 1213 (108) (Burkholderia ambifaria [106) exhibits potent antifungal activity against S. cerevisiae (MIC = 0.15 μg/mL), C. albicans (MIC = 0.15 μg/mL), and A. niger (MIC = 0.15 μg/mL) [107) exhibits potent antifungal activity against S. cerevisiae (MIC = 0.1 μg/mL), C. albicans (MIC = 0.4 μg/mL), and A. niger (MIC = 0.1 μg/mL) [108) exhibits potent antifungal activity against S. cerevisiae (MIC = 2.0 μg/mL), C. albicans (MIC = 31.0 μg/mL), and A. niger (MIC = 2.0 μg/mL) [106) and 1119 (107) containing 2,4-diaminobutyric acid adjacent to the 3-OH-Tyr structure is higher than that of burkholdines 1229 (104), 1097 (105), and 1213 (108) containing Asn adjacent to the 3-OH-Tyr structure [106) and 1119 (107) is significantly higher than hemolytic activity, but the antifungal activity of burkholdines 1229 (104), 1097 (105), and 1213 (108) is equivalent to hemolytic activity [107), assigning an Asn replaced a 3-OH-Asn and attaching a β-xyloside glycosyl portion replaced a α-xyloside glycosyl portion compared to burkholdine 1215 (106), shows weaker activity against C. albicans (MIC = 0.4 μg/mL) and stronger activity against A. niger (MIC = 0.1 μg/mL). Burkholdine 1213 (108), attaching an Asn replaced a 2,4-aminobutyric acid of burkholdine 1119 (107), shows weaker activity against both C. albicans (MIC = 31.0 μg/mL) and A. niger (MIC = 2.0 μg/mL). Burkholdine 1229 (104), assigning a 3-OH-Asn replaced an Asn of burkholdine 1213(108), shows stronger activity against C. albicans (MIC = 12.5 μg/mL) and weaker activity against A. niger (MIC = 12.5 μg/mL). Burkholdine 1097 (105), removing the β-xyloside glycosyl portion of burkholdine 1229 (104), shows the same activity against C. albicans (MIC = 12.5 μg/mL) and stronger activity against A. niger (MIC = 1.6 μg/mL) (Burkholdines 1229 (97 (105)  are isolria 2.2N . Burkhol5 μg/mL) . Burkhol13 (108)  are isolmbifaria . Burkhol5 μg/mL) . Burkhol1 μg/mL) . Burkhol0 μg/mL) . The anttructure . The antactivity . Burkhol6 μg/mL) .109) , C. utilis (MIC = 5 µg/mL), C. neoformans (MIC = 2 µg/mL), S. cerevisiae (MIC = 20 µg/mL), T. mentagrophytes (MIC = 15 µg/mL), T. rubrun (MIC = 3.9 µg/mL), M. gypseum (MIC < 0.12 µg/mL), Blastomyces derniatitidis (MIC < 0.12 µg/mL), and Hormodendrum pedrosoi (MIC = 15 µg/mL) [109) has demonstrated significant potent efficacy in vivo, as evidenced by its ability to effectively treat T. mentagrophytes hyphae infection in guinea pigs. Specifically, a hydrophilic ointment containing 0.5% funiculosin had a cure rate of 97% within 10 days [109) to guinea pigs and rabbits was negligible, as evidenced by the survival of guinea pigs injected intraperitoneally with 500 mg/kg of the compound. However, it should be noted that funiculosin is toxic to mice and rats, with an LD50 of 5–7 mg/kg for both oral and intraperitoneal administration [Funiculosin (109) , a neutrsum THOM , has beesum THOM ,93 and w5 µg/mL) ,94 (Tabl110) and skyrin (111)  inhibits the growth of C. albicans ATCC 10231 (MIC = 6.25 μg/mL), A. niger ATCC 13496 (MIC = 12.5 μg/mL), and skyrin (111) inhibits the growth of C. albicans ATCC 10231 (MIC = 12.5 μg/mL) [112)  (Fusarium oxysporum (N17B) and exhibits potent antifungal activity against C. albicans with a MIC of 0.78 μg/mL [113) inhibits mammalian phosphatidylinositol 3-kinase in vitro and in vivo and membrane-associated phosphatidylinositol 4-kinase of the fungus Schizosacchromyces pombe [114) and B (115) (Microsphaeropsis species RA10-14 collected from the South China Sea [114) and B (115) have great broad-spectrum antifungal activity against C. albicans, Colletotrichum truncatum, Gloeosporium musarum, and Pestalotia calabae (MICs = 1.56–3.13 μg/mL) [116) and C (117) (Amycolatopsis species M39 [116) shows antifungal activity against C. albicans ATCC 10231 (MIC = 10 μg/mL) and S. cerevisiae ATCC9763 (MIC = 5 μg/mL) [117) shows weaker antifungal activity against C. albicans ATCC 10231 (MIC = 25 μg/mL) and S. cerevisiae ATCC9763 (MIC = 20 μg/mL) [118), hakuhybotrol (119), cladobotric acid A (120), cladobotric acid F (121), cladobotric acid E (122), cladobotric acid H (123), and pyrenulic acid A (124) , B (126), C (127), and D (128) , B (126), C (127), and D (128) show antifungal activity against C. neoformans H99 with MIC values of 8 μg/mL, 4 μg/mL, 4 μg/mL, and 8 μg/mL, respectively (Emodin (in (111)  are anths ZH-154 . Emodin 5 μg/mL) . HippolaL) [112)  is a furμM [113)  is a fur78 μg/mL . Wortmanes pombe . Microke B (115)  are isolhina Sea . Microke3 μg/mL) . Macrote C (117)  are macrcies M39 . Macrote5 μg/mL) . Macrote0 μg/mL) . F2928-1 A (124)  are isola FKA-73 . These clentulus . Campafu D (128)  are isolectively .In conclusion, this review provides a comprehensive overview of natural antifungal polyketides encompassing various subclasses such as polyethers, macrolides, xanthones, linear polyketides, anthraquinone, polyphenols, pyridine derivatives, furan derivatives, pyranan derivatives, monophenyl derivatives, macrolactam polyketides, hybrid polyketide non-ribosomal peptides, and other polyketides. Additionally, this review discusses the origin, in vitro and in vivo antifungal activities, structure–activity relationship (SAR), safety profile, mechanism of action, and the impact of structural modifications on the SAR of these polyketides. Previous studies on polyketides have demonstrated the substantial antifungal properties exhibited by certain natural polyketides, such as amphotericin B and caspofungin. This observation suggests that polyketide lead compounds hold considerable potential for the future treatment of fungal infections. Given the remarkable antifungal activities displayed by natural polyketides, this class of compounds has garnered significant interest as a potential therapeutic avenue for fungal infections in the future. Currently, the predominant research emphasis lies in synthesizing novel polyketides, while the clinical utilization of pre-existing polyketide compounds as antifungal medications remains limited. Consequently, further comprehensive and meticulous clinical investigations are imperative to substantiate their efficacy in the future. Moreover, the antifungal properties of unnatural polyketide compounds can potentially be harnessed through combinatorial biosynthesis. Numerous PKSs facilitate the production of primary polyketide compounds, which lack biological activity until they undergo modification by PKS post-modifying enzymes, thereby presenting a promising avenue for exploring new antifungal drugs. Polyketides’ chemical composition and fungicidal properties can be altered by utilizing various post-modification enzymes, including cyclase, aromatase, glycosylase, and halogenase. Unconventional antifungal polyketides have been synthesized by modifying the modules, domains, and subunits of PKSs and employing site-directed mutagenesis techniques. Furthermore, the enhancement of antifungal polyketide production or the acquisition of novel antifungal polyketides can be achieved by combining initiation substrates and elongation units from different hosts and implementing targeted modifications. Only a small number of antifungal natural polyketide compounds have been identified. Many habitats of microorganisms, plants, animals and marine organisms have not been explored, and many antifungal polyketide products urgently need to be discovered. Natural polyketides from microorganisms such as fungi are scarce and difficult to obtain. The solubility, safety, and in vivo bioavailability of natural polyketides should also be considered. Semisynthetic components of natural products will play an important role in developing antifungal candidates in the future.


Passive immunization via maternal antibodies (Ab) is a key mechanism for infant protection in early life. This study analyzed the landscape of passive immunization induced by SARS-CoV-2 infection and/or vaccination by comparing the magnitude and durability of the transferred Ab.Prospective multicenter observational cohort study of SARS-CoV2-infected and/or vaccinated pregnant women and their infants. We collected maternal and cord blood samples at delivery and neonatal/infant samples at delivery, and at 1, 2, 6 and 12 months (mo) of age. RBD and Spike IgG Ab titers were measured by ELISA.10 ng/ml and from infected and vaccinated mothers [4.61 (4.27-4.93)] log10 ng/ml were higher than those from infected mothers [2.20 (0.10-3.30)] log10 ng/ml, p< 0.001. These differences were observed until 6 mo of age, when only 1/13 (7.7%) infant from infected mothers had detectable maternal Ab compared to 14/15 (93.3%) and 8/8 (100%) from the vaccinated, and infected and vaccinated groups, respectively. At 12 mo none of the infants born to infected mothers had detectable maternal Ab, while in the vaccinated group (n=11), 3 (27.3%) had persistent Ab, and 4 (36.4%) suspected infection. At 12 mo in the infected and vaccinated group (n=6) 3 had no Ab, and 3 had suspected infection. The median [IQR] placental Ab transfer ratio was higher in the vaccinated only group (2.94 [1.34-3.74]) than the infected only group (1.19 [0.33-2.52]) p< 0.01. Linear regression analyses demonstrated that the mean RBD Ab transfer ratio for infants from vaccinated vs infected women was 1.76  higher after adjusting for trimester of infection or vaccination. Similar results were observed with Spike Ab.215 mothers  and 174 infants  were enrolled. At birth RBD Ab titers median [IQR] of infants from vaccinated mothers [4.28 (3.48-4.80)] logDemographic and clinical characteristics of the study populationm; median. IQR; interquartile range. N/A; not applicable.Infants from vaccinated mothers demonstrated maternal Ab up to 6 mo with higher titers than infants born from infected mothers. Antibodies generated by vaccination were transferred more efficiently than those generated by infection regardless of the trimester in which occurred.Asuncion Mejias, MD, PhD, MsCS, Astra-Zeneca: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Sanofi-Pasteur: Advisor/Consultant Octavio Ramilo, MD, AstraZeneca: Honoraria|Bill & Melinda Gates Foundation: Grant/Research Support|Janssen: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Honoraria|NIH: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Honoraria|Sanofi: Advisor/Consultant|Sanofi: Honoraria


Candida, Aspergillus, and Pneumocystis spp. in BMT.Rezafungin (RZF) once weekly (QWk) is a next-generation echinocandin in development for treatment of candidaemia and invasive candidiasis (IC) and for prevention of invasive fungal disease caused by The Phase 3 ReSTORE treatment trial (NCT03667690) demonstrated RZF QWk noninferiority to caspofungin (CAS) QD. This sub-analysis evaluated efficacy and safety outcomes in patients identified as immunocompromised (immuno-c) during the trial.As previously described, adults with confirmed candidaemia and/or IC were randomized to RZF QWk (400 mg Wk1 then 200 mg QWk) or CAS QD for ≥14 days (≤4 weeks) with optional oral fluconazole stepdown for CAS. In this sub-analysis, immuno-c patients were those with prior and/or concomitant use of immunosuppressants  and/or medical history ongoing at screening of neutropenia, BMT, SOT, lymphoma or leukaemia.Of 187 patients (mITT population), 90 were immuno-c as defined. Results are for those with data available for analyses: Day 14 global cure—Immuno-c: RZF, 51.1% (23/45); CAS, 55.6% (25/45); –No immuno-c: RZF, 66.7% (32/48); CAS 65.3% (32/49). Day 5 mycological eradication—Immuno-c: RZF, 64.4% (29/45); CAS, 51.1% (23/45); no immuno-c: RZF, 72.9% (35/48); CAS, 71.4% (35/49). Among immuno-c, more had ≥1 AE (96.9%) and ≥1 SAE (64.6%) versus no immuno-c (79.0% and 45.0% respectively).In this sub-analysis of ReSTORE, immune-c status reduced efficacy rates overall but did not change efficacy rate differences between RZF and CAS.