Candida species have evolved intrinsic and acquired resistance to a variety of antifungal medications. The primary goal of this literature review is to summarize the molecular mechanisms associated with antifungal resistance in Candida species. Resistance can be conferred via gain-of-function mutations in target pathway genes or their transcriptional regulators. Therefore, an overview of the known gene mutations is presented for the following antifungals: azoles , echinocandins , polyenes (amphotericin B and nystatin) and 5-fluorocytosine (5-FC). The following mutation hot spots were identified: (1) ergosterol biosynthesis pathway mutations (ERG11 and UPC2), resulting in azole resistance; (2) overexpression of the efflux pumps, promoting azole resistance ; (3) cell wall biosynthesis mutations , conferring resistance to echinocandins; (4) mutations of nucleic acid synthesis/repair genes , resulting in 5-FC resistance; and (5) biofilm production, promoting general antifungal resistance. This review also provides a summary of standardized inhibitory breakpoints obtained from international guidelines for prominent Candida species. Notably, N. glabrata, P. kudriavzevii and C. auris demonstrate fluconazole resistance.Candidiasis is a highly pervasive infection posing major health risks, especially for immunocompromised populations. Pathogenic  Candida genus . SteCandida strains do not inherently contribute to antifungal resistance ; therefore, mutations affecting their efficacy may differ. For example, ERG11 point mutations K128T and Y132H may affect the ability of fluconazole or voriconazole molecules to enter or bind the target active site. Mutations in other gene sequences can also confer resistance, such as the G464S mutation, which affects haem coordination due to its location near a key cysteine residue . These muptake control 2 (Upc2) regulates most of the genes in the ergosterol biosynthesis pathway on some level  dienol) by downstream enzymes  . When itida spp. ,114,115.albicans . Q139A ssistance  99]..Candida C. albicans ..C. albicC. albicans genome, evidence of transporter overexpression in resistant clinical isolates is currently limited to ABC-Ts CDR1 and CDR2 and MFS-Ts MDR1 and PDR16  and SNQ2 and MFS-T genes QDR2, FLR1 and PDR16 has been cited . F. F156]. glabrata ,159,160.esistant ,78. For  members .C. albicans fungal cells only require one mutant allele for resistance to be conferred across the three echinocandins [C. albicans strains, which may limit population spread for these mutants under non-echinocandin treatment conditions [Candida species [Significant GOF mutations in the FKS1 gene sequence in clinical isolates have been described, particularly in the hotspot regions of 637–654 and 1345–1365  74,157,,167,168.nditions . This is species .N. glabrata isolates  and S663P (FKS2) mutations and indicated that genotyping of these regions is an accurate indicator of resistance to echinocandins [Candida strains. As a result, these validated gain-of-function mutations are one example of a potential biomarker for antifungal resistance. PCR assays were developed to detect these mutations, which has aided in treatment, although there is still a lack of a timeliness in achieving this result [Bienvenu et al. (2019) highlighted the ocandins . A relats result ,174. Recs result ,176. Oves result .fks genes can also affect echinocandin resistance. In particular, point mutations in transcription factor PDR1 have been found in numerous resistant N. glabrata isolates  and FUR1 (uracil phosphoribosyltransferase (UPRT)) [C. albicans and other Candida species is the FCA1 gene [The biosynthesis of nucleic acids (DNA and RNA) and subsequent protein synthesis in pathogenic fungi can be targeted with nucleoside analogue 5-fluorocytosine (5-FC). As a prodrug, it requires activation within the fungal cell via metabolism by the pyrimidine salvage pathway . Then, i (UPRT)) . The FCYCA1 gene ,190. TheCA1 gene .Saccharomyces cerevisiae [S. cerevisiae yeast cells [Candida samples are presented in Resistance to 5-FC could arise with mutation or loss of any of the three key enzymes , as discovered in model organism yeast revisiae ,193. Increvisiae ,83. Kernst cells . The nonCandida biofilms have intrinsic antifungal resistance [C. albicans is most often associated with biofilm formation [Candida species that can form biofilms include C. auris, N. glabrata, C. dubliniensis, P. kudriavzevii, C. parapsilosis and C. tropicalis [Yeast cells can grow freely/planktonically or develop an extracellular matrix (ECM) to attach to and form a highly organized community of microbial cells ,204. Thesistance . The arcsistance ,210,211.ormation ,213,214.opicalis ,215,216.C. albicans with biofilms can be increased by up to 1000-fold compared to planktonic cells [C. albicans biofilms compared to planktonic cells demonstrated an approximately 10-fold increase in amphotericin B resistance [Over the course of antifungal treatment, a biofilm can develop and enable yeast cells to become more resistant ,215. Numic cells . Alternasistance . One stusistance .Multiple features of biofilms enable antifungal resistance aside from genetic mutations that typically drive resistance in planktonic cells, which adds to the complexity of treating this type of infection . These iCandida infections with biofilms [Candida biofilms, and multidrug resistance has been cited, including against azoles, echinocandins and polyenes [One of the main ECM components is β-1,3 glucan, which is synthesized by echinocandin target gene FKS1 . The polbiofilms . Supplembiofilms ,226. Furpolyenes . Other tpolyenes ,229. Thepolyenes ,229.Candida strains associated with biofilm formation and resistance to antifungal treatment may have similar key genes implicated in planktonic resistant isolates. Changes in the expression of ergosterol biosynthesis pathway genes and in biofilm membrane composition appear to be linked to subsequent azole and polyene resistance [Candida exposed to amphotericin B, in agreement with previous studies [sistance ,232,233. studies ,230. The studies .Candida strains [Candida infections of this type. This suggests that echinocandins could be a preferable treatment choice over azoles [Candida infections and biofilms is provided in [Drug efflux pumps CDR1, CDR2 and MDR1, which are associated with azole resistance, may also be upregulated in biofilm-associated  strains ,239,240.r azoles ,237,241.r azoles ,243. Impr azoles . Therefor azoles . A detaivided in .Candida species. Currently, PCR-based methods using fungal cultures are still the first option for species identification and detection of antifungal resistance in individual strains [Candida species to modify the treatment course has been well documented. For example, two patient cases of candidemia presenting with fluconazole-resistant C. albicans strains were successfully treated with amphotericin B, despite higher cost and risk of greater side effects [With antifungal resistance being a continued problem, there is a need for the development of quick and reliable molecular diagnostic tests that detect organisms with intrinsic and/or secondary resistance due to the genetic mechanisms presented in this review . Additio strains ,167. The effects .C. albicans clinical isolates using microarray analysis identified almost two hundred genes (n = 198) that were differentially expressed [Resistance can be acquired through a dynamic combination of numerous point mutations and other genetic or transcriptional alterations. A large-scale comparison between matched fluconazole-resistant and -susceptible xpressed . In resixpressed . This hiCandida species need to be established. This review presents numerous possible gene mutation biomarkers that have been associated with antifungal resistance in clinical isolates from studies worldwide. It is important to note that different non-synonymous substitutions and other genetic alterations may result in similar genetic effects. In this scenario, assessing the mRNA overexpression of ERG11 may be a good biomarker for antifungal resistance, especially azoles. Pfaller et al. (2006) suggested that a test that can identify resistant Candida strains with high MICs would be more clinically useful than predicting susceptibility according to low MICs [To develop a dependable point-of-care (POCT) diagnostic assay for antifungal resistance, reliable biomarkers in low MICs .Candida clinical isolate samples could elucidate other drug targets and support the development of alternative antifungal treatments [The emergence of new technologies, including next-generation sequencing, CRISPR and isothermal amplification-based detection assays, has enabled progress in the development of reliable assays to detect pathogenic nucleic acid profiles ,251,252.eatments .Candida. The identification of fungal surface proteins with immunogenic properties and, ideally, no additional side effects in humans could be used to produce an mRNA vaccine. The proactive immune response elicited when these proteins are expressed within the human body could protect the individual in defending against candidiasis infections in the future. The recent progress in mRNA vaccine technology for viruses has made this an important and promising option to address fungal infections [Also, the development of antifungal vaccines would be aided by adequate characterization of pathogenic fections .Candida genus and their differing responses to antifungal treatments. Notably, N. glabrata, C. krusei and C. auris are all highly fluconazole-resistant. The ability to identify these fungal species at the time of candidiasis diagnosis could improve treatment selection by prescribing echinocandins, polyenes or some combination thereof where appropriate. Overall, understanding the mechanisms of antifungal resistance in various pathogenic Candida can aid in developing POCT assays to identify resistant strains and positively contribute to clinical outcomes in the management of candidiasis.Furthermore, this review of antifungal resistance highlights the diversity of members of the


The temperature cooking of meat such as grilling and smoking contribute to the formation of mutagenic compounds including heterocyclic amines and polycyclic aromatic hydrocarbons.Heme iron in red meat is involved in the formation of N-nitroso compounds and lipid peroxidation products in the digestive tract.Fatty red meat is involved in the production of secondary bile acids by the bacteria of the gut microbiota.Excessive consumption of red and processed meat has been associated with a higher risk of developing colorectal cancer. There are many attempts to explain the risk of colorectal cancer associated with the consumption of red and processed meat:Many of the products formed are genotoxic and can cause DNA damage and initiate carcinogenesis of colorectal cancer. Various mechanisms contributing to their genotoxic role have been established in human and animal studies. In addition, there is increasing evidence that compounds formed from red and processed meat interact with the gut microbiota in colorectal cancer pathways. Although several early studies in animals and humans suggest a direct causal role of the gut microbiota in the development of colorectal cancer, the links between diet, gut microbiota, and colonic carcinogenesis are largely associations rather than proven causal relationships. Various biological mechanisms, including inflammation and oxidative stress can lead to DNA damage, gut dysbiosis, and therefore increase the risk of colorectal cancer. Dysbiosis of the gut microbiota may increase the risk of colorectal cancer through dietary component promotion of colonic carcinogenesis. In this paper, we review and update current knowledge about the relationships between red meat consumption, gut microbiota, and colorectal cancer. Colorectal cancer (CRC) is the third most common cancer, diagnosed in both men and women, with an estimated 1.9 million new cases and 935 000 deaths reported in 2020, worldwide [Rectal and colon cancers related deaths are estimated to be in 60% and 71.5% respectively in 2035), making CRC a worldwide public health concern . EpidemiOf 478,040 participants enrolled in the European Prospective Investigation into Cancer and Nutrition (EPIC) trial who were prospectively followed between 1992–1998, 1,329 CRC cases were detected and an association with red and processed meat consumption was observed . RecentlIn this paper, we review and update current knowledge regarding the interaction between the gut microbiota and CRC, the impact of red meat consumption on the gut microbiota, and the contribution of red meat consumption to the pathogenesis of CRC.Numerous epidemiological and scientific studies suggest that the risk of CRC is increased by red and processed meat consumption ,28,29. AIngestion of red meat rich in fat and excessive secretion bile acid (BA) have been cited in several animal and human experimental studies as one of the factors promoting the increased risk of CRC. Primary BAs, including cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized by the liver following the digestion of dietary lipids in the stomach to emulsify fats. After lipid digestion, most primary BAs are deconjugated and reabsorbed (enterohepatic recycling). Still, a small amount may pass into the colon where they are transformed into secondary BAs (deoxycholic acid and lithocholic acid) by colonic bacteria by enzyme 7α-dehydroxylation as shown in HCA are formed from products in processed meat  when cooked at high temperature . The forHughes et al. reported, for the first time in 2001, a significant excretion of NOC in the feces of volunteers who consumed large amounts of red meat . NOC areThis evidence suggests that NOC, formed from red and processed meat, can promote the development of CRC by inducing mutations in key tumor suppressor genes, including APC and TP53, and oncogenes such as KRAS.The carcinogenic role of heme iron in red and processed meats is supported by numerous epidemiological and experimental studies ,65. ThreThe mechanism of trimethylamine–N-oxide (TMAO)-mediated CRC carcinogenesis in humans and animals has been examined in several studies . TMAO isH. pylori infection in the stomach [The inescapable role of the gut microbiota in the generation of TMAO was demonstrated by a study of antibiotics given to human subjects for one week to eliminate the gut microbiota . The eli stomach –84. The  stomach . It may  stomach . In viewEscherichia, Pseudomonas, Proteus, Klebsiella, and Neisseria) is low in humans, but may increase with a diet rich in nitrates or nitrites [E. coli during an intestinal inflammatory response mediated by nitrate compounds [The interaction between compounds formed from red meat directly or indirectly after its ingestion, including N-nitroso compounds (NOC), heterocyclic amines (HCA), polycyclic aromatic hydrocarbons (PAH), heme iron, bile acids (BA), Trimethylamine-N-oxide (TMAO) and the gut microbiota has been reported in numerous animal and human studies as shown in nitrites ,87–89. Nnitrites . Nitratenitrites –93. In mompounds . Previouompounds ,95. A stompounds . Althougompounds –32. Therompounds ,96,97.E. coli multiplication and the formation of NOCs are responsible for promoting inflammation or the development of CRC. Exposure of mouse models to PAHs also causes change in the composition of the gut microbiota after colonic inflammation. In humans, PAHs and HCAs altered the volatile profile of the fecal microbiota and their metabolite activities after high exposure of the gut microbiota over 24 hours to these substances [It is not known, however, whether or not the combined action of nitrate-induced bstances ,98,99.Eulonchus halli) through their beta-glucuronidase and glycerol/diol dehydratase activities that convert HCA to HCA-M1 [Certain bacteria may reduce the risk of CRC associated with HCA consumption in the gut microbiota , Escherichia coli (E.coli), Streptococcus bovis (S. bovis), Enterococcus faecalis , Clostridium septicum (C. septicum), Fusobacterium nucleatum (F. nucleatum), Enterotoxigenic Bacteroides fragilis (ETBF) and Streptococcus gallolyticus  were suspected as factors promoting CRC [Whole genome sequencing of gut microbial species has allowed researchers to study the microbial communities that colonize colonic tumors as well as non-tumor colonic sites and to characterize individual oncogenic microbiomes . The proting CRC ,111. To ting CRC . Driver Klebsiella pneumonia and Proteus mirabilis could be treated as key pathogens of inflammatory bowel disease, and the role of ETBF in CRC. This model may provide new insights to review the potential role of gut pathogens in the initiation and progression of related disorders.The other model proposed is the keystone model, which supports the role of a key pathogen in the process of dysbiosis associated with a given disease . This hyF nucleatum in CRC carcinogenesis has been reported through studies [F. nucleatum infiltrates the colonic tumor through its adhesin (FadA), selectively binding to E-cadherin and activates the β-catenin signaling pathway, inducing inflammatory responses allowing CRC progression [F. nucleatum also inhibits T cell and natural killer cell activity through another adhesin, fibroblast activation protein 2 (Fap2), which binds to T cell immunoglobulin and the ITIM domain [F. nucleatum to infiltrate the colonic tumor by binding to the carbohydrate moiety D-galactose-β (1–3)-N-acetyl-D-galactosamine , which is overexpressed in CRC cells [F. nucleatum-enriched colonic tumors [F. nucleatum adhesin Fap2, which induced the secretion of the pro-inflammatory cytokines, IL-8 and CXCL1 [F. nucleatum in Apc Min/+ mice and in human CRC cell lines was indicated by Nuclear factor-kappa B (NF-κB) activation which in turn induced miR21 gene expression promoting inflammatory responses [F. nucleatum was significantly increased in patients with early stage CRC and that the presence of F. nucleatum in CRC tissues is associated with a poor prognosis of the disease [The potential role of  studies . Analysi studies . F. nuclgression . F. nuclM domain . Fap2 adRC cells . Interlec tumors . A recennd CXCL1 . The caresponses . Another disease ,123,124.B. fragilis toxin (BFT) that cleaves E-cadherin on colonic epithelial cells resulting in disruption of the colonic barrier [E. faecalis and ETBF copy number were significantly higher in CRC tissue samples compared to the no CRC group [Several studies have reported the link between ETBF and CRC and its use as a potential biomarker in the diagnosis of CRC –127. ETB barrier . The dis barrier ,130. A rRC group . ETBF alRC group ,133. ETBRC group .S. gallolyticus is essential for its attachment to human mucus-producing epithelial cells (135). Interestingly, pil3 binds both to the human mucin 2 (MUC2), which predominates in healthy colonic tissue, and to mucin 5AC (MUC5AC), which is overexpressed in cancerous colonic tissue [S. gallolyticus is facilitated by its binding to the mucin MUC2, while it’s binding to MUC5AC gives a growth advantage to bacterial species of the gut microbiota in the tumor microenvironment [S. gallolyticus in the presence of colonic tumors. Colonic epithelial cells from CRC patients showed high NF-κB gene expression in the S. gallolyticus-positive group compared with the S. gallolyticus-negative group [S. gallolyticus seropositive CRC patients, both of which are products of NF-κB activity [The pilus 3 (pil3) of al cells . Interesironment . This heve group . NF-κB pactivity .E. coli, is an anaerobic gram-negative commensal bacterium, commonly found in the intestinal microenvironment. Some groups of E. coli called pathotypes belonging mainly to the B2 and D phylogroups have been identified as potentially oncogenic and pro-inflammatory [E. coli groups to CRC risk. Enrichment in B2 and D phylogroups was reported in CRC patients compared to control subjects (without CRC) [E. coli enriched colorectal tissue, compared with 42% of 24 control biopsies without CRC [E. coli in CRC patients compared to healthy subjects [E. coli groups and CRC risk has been proposed through several studies. Nevertheless, the mechanism involved is very poorly understood to date. But according to studies, pathogenic E. coli strains producing cyclomodulins, and toxins are responsible for induction of DNA damage and cell cycle disruption in eukaryotes [E. coli was observed in CRC patients compared to healthy subjects [E. coli to show transient DNA damage [E. coli strains induced an acceleration of tumor development compared to uninfected control mice or mice infected with a non-colibactin-producing mutant of these strains, or animals infected and treated with molecules inhibiting colibactin synthesis [E. coli strains is known to have DNAase activity resulting in DNA double strand breaks, cell division arrest and inhibition of cell apoptosis [ammatory . Severalout CRC) . Indeed,hout CRC . In addisubjects –144. Thekaryotes ,139,145.subjects . These gsubjects ,147. Thesubjects –150. CulA damage . AccordiA damage ,152. In ynthesis ,144,153.ynthesis . CIF proynthesis . CNF-1 aynthesis ,157. TheE. coli, E. faecalis is part of the facultative anaerobic Gram-positive commensal flora and does not appear to be offensive to humans. However, studies have shown enrichment of CRC patients’ fecal samples and tumor tissues by E. faecalis compared to those healthy individuals [E. faecalis species was significantly lower in obese patients than in non-obese patients with CRC [E. faecalis was relatively higher in obese subjects than in non-obese subjects. This study demonstrated that a reduced presence of E. faecalis may be associated with obesity-related CRC carcinogenesis. Another recent study conducted on 256 fresh frozen CRC tissues detected E. faecalis in 193 of the 256 CRC tissues [E. faecalis bacteremia was observed in an 86-year-old white male during a secondary gastrointestinal hemorrhage with confirmation of colorectal adenocarcinoma by colonoscopy [E. faecalis was able to promote and maintain colitis in Il10−/− or Il10 gene deficient mice with induction of rectal dysplasia and carcinoma [E. faecalis, thereby activating Smad signaling. Interestingly, these mice lost toll-like receptor (TLR2) expression with NF-κB-dependent pro-inflammatory gene inhibition, in contrast to Il10−/− mice that failed to inhibit TLR2 receptor-mediated pro-inflammatory gene expression in intestinal epithelial cells upon colonization by E. faecalis. (163) Blood isolates of E. faecalis have also been associated with the production of extracellular superoxide (O2−) and hydrogen peroxide (H2O2) in the intestine [E. faecalis is able to induce DNA damage in vivo in colonic cells in rats. Extracellular infection of mammalian cells by E. faecalis can result in the production of superoxide (O2−) leading to overexpression of COX-2 in macrophages and promotes chromosomal instability in primary colonic epithelial cells [E. faecalis is able to polarize colonic macrophages into an M1 phenotype, which in turn induces aneuploidy and chromosomal instability in colonic epithelial cells commonly found in cancers [E. faecalis exerts its impact on CRC.Like ividuals ,159. A rwith CRC . Similar tissues . E. faeconoscopy . E. faecarcinoma . Intestintestine ,165. Theal cells . Similar cancers . These dS. bovis and CRC in 1951 [S. bovis strain was isolated from stool samples of 35 out of 63 CRC patients compared to 11 out of 105 control individuals without CRC [S. bovis and CRC [S. bovis induced an overexpression of pro-inflammatory mediators, notably IL-8, COX-2 and PGE2 [S.bovis.Azoxymethane-treated rats confirmed the release of pro-inflammatory mediators after infection with S. bovis, which explains the increase in the number of aberrant crypts. Interestingly of the azoxymethane-treated rats, three of the six treated rats developed polyps during S. bovis infection, whereas no polyps were detected in the uninfected azoxymethane treated rats. Hyperproliferative crypts were also detected in azoxymethane-treated rats with S. bovis infection, demonstrating the involvement of this bacterium in CRC carcinogenesis [S. bovis-infiltrated tissues compared with non-infiltrated tissues. Similarly the expression of these mRNAs was elevated in tumor tissues compared to non-tumor tissues. The pro-inflammatory profile of S. bovis may increase the risk of CRC development and progression.McCOY WC et al. first established the link between  in 1951 . Subsequhout CRC . Subsequ and CRC ,171. Accand PGE2 . Other aogenesis . Throughogenesis . StudiesH. pylori is a gram-negative bacterium that preferentially infects and colonizes gastric tissue in humans. Although most infected individuals remain asymptomatic, H. pylori can induce chronic inflammatory responses increasing the risk of gastric ulcer, and adenocarcinoma of the stomach [H. pylori, it has been shown that its toxicity can extend outside the stomach. The link between H. pylori infection and CRC remains controversial with studies showing a strong association with a high prevalence of H. pylori infection in patients with colonic adenomas and carcinomas [H. pylori infection and an increased occurrence of CRC [H. pylori infection and CRC. Despite the ambiguity between some studies on the link between H. pylori infection and CRC, others have attempted to explain the molecular mechanism underlying the potential association between H. pylori infection and CRC with some supporting hypotheses such as toxin release, dysbiosis and chronic inflammation. For example, significant gastrin secretion mediated by H. pylori infection was associated with increased expression of COX-2 and anti-apoptotic B-cell lymphoma 2 (BCL2) protein compared to pro-apoptotic BCL2 Associated X(BAX) protein resulting in decreased apoptosis in CRC [B. fragilis and E. faecalis. Other mechanisms of CRC carcinogenesis mediated by H. pylori infection have been proposed. These include the production of ROS and reactive nitrogen species (RNS) that can lead to DNA damage, which could promote CRC carcinogenesis [H. pylori strains toxicities’ vary from patients. For example, strains that carry the virulence factor cytotoxin-associated gene A (CagA) are more toxic than those without, and patients with these strains have an increased risk of developing gastric cancer and CRC compared to those without [H. pylori strains can induce production of pro-inflammatory mediators such as TNF-α, IFN-γ, IL-1β, IL-6 and IL-8 by infected cells [H. pylori give it a pro-inflammatory property that can promote gastric and CRC carcinogenesis. stomach . Despitercinomas ,176. Whircinomas ,178. Mane of CRC –181. Sims in CRC . High gas in CRC . This dyogenesis . In addi without . The Vaced cells ,187. TheC. septicum is a Gram-positive, anaerobic, spore-forming bacillus, which is not normally present in the intestinal flora, but can cause direct and spontaneous infections in the gut.C. septicum infection and CRC [C. septicum spores [C. septicum were associated with malignancy. The alpha-toxin-producing C. septicum group is associated with the release of TNF-α after activation of the mitogen-activated protein kinase (MAPK) pathway, which has been shown to be dysregulated in cancers [C. septicum may promote carcinogenesis. However, despite available data, no direct link between C. septicum and CRC has been defined to date.This bacterium produces alpha-toxin, a virulence factor which is both lethal and hemolytic in mice . Several and CRC –191. Thim spores . Eighty  cancers –201. ThiE. coli, S. gallolyticus, and F. nucleatum are frequently initiators of colonic carcinogenesis through virulence factors and responsible for CRC progression after their infiltration into the tumor microenvironment. Animal and human experimental studies also strongly support the evidence of diet being a major risk factor for CRC. More longitudinal clinical studies are needed to confirm and better understand the mechanisms underlying the diet-mediated disruption of gut microbiota in humans and establish the direct cause and impact that dysbiosis has on the initiation and progression of CRC.This review examined current knowledge about risk factors of CRC carcinogenesis. Red and processed meat consumption and its interaction with the gut microbiota are found to be major associated factors. The CRC-associated gut microbiota is made of pro-inflammatory or pro-carcinogenic bacteria and opportunistic pathogenic bacteria that enrich the tumor microenvironment by promoting disease progression. Bacteria such as


Pholidota Lindl. ex Hook. is economically important as some species has long been used in traditional medicine. However, the systematic status of the genus and intergeneric relationships inferred from previous molecular studies are unclear due to insufficient sampling and lack of informative sites. So far, only limited genomic information has been available. The taxonomy of Pholidota remains unresolved and somewhat controversial. In this study, the complete chloroplast (cp.) genomes of thirteen Pholidota species were sequenced and analyzed to gain insight into the phylogeny of Pholidota and mutation patterns in their cp. genomes.The orchid genus Pholidota cp. genomes exhibited typical quadripartite circular structures, with the size ranging from 158,786 to 159,781 bp. The annotation contained a total of 135 genes in each cp. genome, i.e., 89 protein-coding genes, 38 tRNA genes, and eight rRNA genes. The codon usage analysis indicated the preference of A/U-ending codons. Repeat sequence analysis identified 444 tandem repeats, 322 palindromic repeats and 189 dispersed repeats. A total of 525 SSRs, 13,834 SNPs and 8,630 InDels were detected. Six mutational hotspots were identified as potential molecular markers. These molecular markers and highly variable regions are expected to facilitate future genetic and genomic studies. Our phylogenetic analyses confirmed the polyphyletic status of the genus Pholidota, with species grouped into four main clades: Pholidota s.s. was resolved as the sister to a clade containing species of Coelogyne; the other two clades clustered together with species of Bulleyia and Panisea, respectively; species P. ventricosa was placed at the basal position, deviated from all other species.All examined thirteen Pholidota based on plastid genomic data. These findings contribute to a better understanding of plastid genome evolution of Pholidota and provide new insights into the phylogeny of Pholidota and its closely related genera within the subtribe Coelogyninae. Our research has laid the foundation for future studies on the evolutionary mechanisms and classification of this economically and medicinally important genus.This is the first study to comprehensively examine the genetic variations and systematically analyze the phylogeny and evolution of The online version contains supplementary material available at 10.1186/s12870-023-04233-8. Pholidota Lindl. ex Hook. is an orchid genus, which was described in 1825 by W.J. Hookers in his Exotic Flora. As current circumscribed, Pholidota belongs to the subtribe Coelogyninae, tribe Arethuseae of the subfamily Epidendroideae [droideae .Pholidota is of great economic and medicinal importance. Several species of this genus possess a broad range of medicinal properties and have been commonly used as folk herbal medicine for various medicinal purposes in China and India for many centuries [Pholidota [Coelogyne, Pholidota and other related genera are not monophyletic and the relationships among them still remain elusive [Panisea has been usually recognized as a natural group, we find that some key characteristics for group identification such as basally sigmoid or shallowly saccate lip [Panisea and Pholidota. The taxonomic complexity of these taxa is largely caused by high morphological diversity at both intrageneric and intergeneric levels and homoplasy of the major diagnostic characters. The taxonomic complexity of these groups makes generalizations difficult. Quite a few questions remain concerning these taxa including whether they should be considered distinct genera and how to circumscribe them.enturies –7. Thereholidota , 2. Prev elusive . Likewiscate lip  are highPholidota have been neglected. So far, information about Pholidota with regard to the genomic characteristics of the chloroplast genomes has been limited. The phylogenetic relationships between Pholidota and its closely related genera within Coelogyninae remain unclear. In the current study, we focused on (1) analyzing the thirteen cp. genome structural characteristics of Pholidota, (2) elucidating the genetic diversity and developing optimized markers for discriminating Pholidota species, (3) evaluating the phylogenetic position of Pholidota and discovering the most probable intra- and intergeneric relationships among Pholidota and its allies using chloroplast genome alignments.Recent advances in molecular genomics and bioinformatics, particularly next-generation sequencing approaches present a phylogenomic framework for charting the diversity and evolution of angiosperm. Compared with the nuclear genome, the chloroplast genome has distinct features, e.g., maternal inheritance, high conservation and appropriate polymorphism. These properties make genetic polymorphism of the plastome a suitable source of molecular markers for a range of genetic and phylogenetic studies at different taxonomic levels in angiosperms –11. It iPholidota cp genomes were provided in Fig. rpl2, rpl23, rps7, rps12, rps19, ycf2, ycf15, and ndhB), four rRNA genes , and eight tRNA genes  were present in two copies. In all the plastomes of these species, ycf1 gene was found to extend from IRa into the SSC region, and left a truncated copy at the junction of IRb/SSC. The rps12 gene in Pholidota plastomes was arranged in a trans-spliced state, with 5’-end exon located in the LSC region and two 3’-end exons located in IR regions. Among all identified genes, eleven protein-coding genes  and eight tRNA genes  each contained two exons, while the other four protein-coding genes  each contained three exons.The graphical genome maps of the newly sequenced Pholidota cp. genomes were calculated  values of 13 Pholidota species. In total, 955 repeats of at least 30 bp long per repeat unit were detected. We categorized these repeats into three types: tandem, dispersed and palindromic. Totally, our analysis identified 444 tandem repeats, 322 palindromic repeats and 189 dispersed repeats in the cp. genomes  and minimum number (25) of tandem repeats were detected in P. missionariorum and P. chinensis, respectively. Meanwhile, P. ventricosa cp. genome had the highest frequency of palindromic repeats (33), whereas the lowest palindromic repeats (17) were detected in P. cantonensis cp. genome. Tandem repeats were found to be highly abundant and frequently dispersed in these genomes. As shown in Fig. Repetitive DNA sequences or repeats refer to homologous DNA fragments that occur as a multiple copy of nucleic acids in the genomes, which are the major components of eukaryotic genomes and considered to play an important role in genome stability and structural variation , 16. In mes Fig. . The numPholidota chloroplast genomes using the Perl script MISA detected 525 microsatellites in four main types, namely mono-, di-, tri- and tetra-nucleotide repeats. A comparison of total SSRs for each species is shown in Fig. P. cantonensis to 48 in P. longipes across the 13 cp. genomes  was located in the cp. genome of P. ventricosa , followed by SSC region (10.64–22.92%), while a minority (4.17–12.5%) occurred within the IR regions , also known as microsatellites or short tandem repeats, refer to short tandemly repetitions of 1–6 base pairs. The SSR analysis of the osa Fig. . In addions Fig. .P. longipes cp. genome as a reference. In total, 22,464 mutations were identified, including 13,834 SNPs and 8,630 InDels  and the minimum number was found in the P. niana cp. genome (233). Similarly, the maximum number of InDels was also detected in P. ventricosa (916), and the lowest was observed in P. niana (264). The average numbers of SNPs and InDels were 1,064 and 267, respectively. Most of Pholidota species have 1,100 to 1,500 SNPs and 280 to 400 InDels. However, the SNPs in the cp. genomes of P. niana (233) and P. protracta (391) were significantly fewer than those in the other species. Similarly, fewer than 160 InDels were discovered in P. niana (62) and P. protracta (153)  and DNA insertions-deletions (InDels) are useful polymorphic markers for analysis of genetic diversity and genetic mapping. In this survey, we determined these genetic variants based on a comparison of 13 cp. genome alignments with the 3) Table . The lowThe frequency and density of SNP/InDel loci identified in the genomic regions varied across the thirteen cp. genomes  to 5,588 bp (P. yunnanensis). At the SSC/IRa border, the ycf1 gene extended into the SSC region, at varying lengths ranging from 4,367 bp in P. missionariorum to 4,568 bp in P. yunnanensis. The truncated copy of ycf1 was largely located in the IRb region, with its one end extending into the SSC region, ranging from 7 bp (P. longipes) to 25 bp (P. protracta). In contrast, ndhF was mainly situated in the SSC region, partially overlapping with the duplicated ycf1 gene. The ndhF gene showed the same length of 2,258 bp in all species, whereas the portion located in the IRb region varied in length from 53 bp in P. protracta to 71 bp in P. longipes. The distance between rps19 and LSC/IRb border was 47 bp in P. cantonensis, whereas 167 bp in P. chinensis. The length from rpl22 to the LSC/IRb border was 43 bp in P. articulata and P. chinensis, in contrast to 73 bp in P. leveilleana and P. yunnanensis. On the other side of the IRa/LSC boundary, psbA gene was found in the LSC region of all genomes but was located 84 bp (P. missionariorum) to 109 bp  apart from the IRa/LSC border.Although the length of the IR regions varied little across the thirteen Pholidota cp. genomes showed obvious changes at the IR/SC boundaries and adjacent genes. Two genes ycf1 and ndhF located at the IR/SSC junctions were found to be highly variable. The results also showed that some differences exist in the non-coding intergenic regions (rpl22-rps19-psbA).In general, the Pholidota cp. genomes were compared and plotted using mVISTA by aligning these cp. genomes with the annotated P. longipes cp. genome as a reference  was measured by DnaSP within 600-bp windows , ndhF-rpl32 (0.0758), rpoB-trnC-GCA (0.0630), rps11-rpl36 (0.0624), and two plastid genes ycf1 (0.1174), ndhA (0.0624) within the coding regions. Among these regions, ndhF-rpl32 was located at the IRb/SSC junction, ycf1 crossed the SSC/IRa border. ndhA was situated in the SSC region, whereas three of six  were located in the LSC region  higher than 0.06  and Bayesian inference (BI) methods. In addition to the 13 newly sequenced genomes, three published cp. genomes of Pholidota and four published cp. genomes from other genera Bulleyia, Coelogyne and Panisea were also retrieved for analyses. Two species of genus Pleione were selected as outgroups based on previously reported relationships [To determine the phylogenetic positions of ionships . The ML Pholidota were generally separated into two main clades, namely clades A and B  containing most other sampled Pholidota species was further divided into three well-supported lineages, designated as subclades I, III, and IV that were found each related to the other genera of the subtribe Coelogyninae. Of the three subclades, subclade I consists of P. imbricata and P. pallida, forming the sister clades to P. articulata, and P. chinensis. Subclade II, which includes two sampled species of Coelogyne , was strongly supported as the sister to subclade I . Within clade III, an accession of Panisea uniflora, fell within the lineage with accessions of P. longipes, P. niana, and P. protracta, constituting a strongly supported monophyletic group , with the former two as the closest sisters. Clade IV consisting of monotypic genus Bulleyia, with its sole representative, B. yunnanensis, together with six accessions of Pholidota, formed a robust monophyletic lineage . A small clade with accessions of P. cantonensis and P. missionariorum was placed as successive sisters to the clade with accessions of P. leveilleana, P. wenshanica and two accessions of P. yunnanensis .The sampled species of d B Fig. . As the Pholidota plastomes presented the typical quadripartite structure of angiosperms and maintained a high degree of consistency among species  that aggregated in these regions. Interestingly, P. cantonensis, a small sized, cool to cold growing lithophytic or epiphytic species endemic to southern China, had a smallest chloroplast size , but a highest GC content (37.47%). This phenomenon has been reported for other orchid species [All the newly assembled ies Fig. . Generalies Fig. . The GC  species , 18. Hig species .Pholidota species were similar. The consistent utilization mode suggests the high conservation of Pholidota plastomes. In particular, these cp. genomes displayed a preference toward A/U-ending synonymous codons. This codon usage pattern was also found in other AT-rich angiosperms [Codon usage bias (CUB) is a unique property of most genomes and varies greatly within and between organisms. It is expected to potentially influence genome evolution and widely used to understand the genetic and evolutionary patterns among different species , 21. Theiosperms –24. FurtPholidota plastomes was similar. However, slight variations of the border positions between the IR and SC regions were still detected  of this gene within IRs. In all these cp. genomes, the ycf1 pseudogene overlapped with the ndhF gene in the IRb/SSC junction, creating various fragment lengths at the IRb region. Previous studies have shown that the stability of the IR/SC boundary regions is mainly correlated with the transformation of gene ndhF and/or ycf1 [Pholidota species. These changes were mainly associated with the different positions of ndhF and ycf1, together with the genes rpl22, rps19 and psbA adjacent to LSC/IR and SSC/IR borders. The distribution and locations of gene types in these regions were highly consistent with previous studies for other orchid species of the subtribe Coelogyninae [In general, the IR regions were relatively more conservative than the LSC and SSC regions and gene content of the IR borders among ted Fig. . For all/or ycf1 –28. We fogyninae .Pholidota varied in size from 158,786 bp (P. cantonensis) to 159,781 bp (P. articulata and P. protracta). The plastome size variation is usually attributed to the expansion and contraction of IR/SC boundary regions [Pholidota plastomes correspond to the IR/SC boundary shifts, which likely provide insights into their genome variation and evolutionary information. Various IR/SC boundary shifts are common evolutionary events and have a major impact on the plastome evolution of land plants [Despite their conservative structural organization, the cp. genomes of  regions , 29, 30.d plants . It has d plants , 33.Pholidota plastomes . Secondly, the intergenic regions  with relatively high variability (> 0.07) were identified, with the former located in the LSC, and the latter located in the IRb/SSC border. Moreover, within the noncoding regions, highly divergent regions also included rpoB-trnC-GCA and rps11-rpl36  analysis of  Pi = 0.14. SecondPholidota was classified into nine sections based on morphological traits. Gravendeel et al. [Pholidota in the “Coelogyne s.s. clade” and considered it to be closely related to the genus Coelogyne. The phylogeny of Pholidota has not been well studied and previous molecular studies based on a few molecular markers included only two samplings of Pholidota species. The phylogenetic placement of Pholidota and its phylogenetic relationship between the genus and other genera remain unclear. This study provides the first phylogeny of Pholidota inferred using the complete cp. genome data and proposes a robust phylogenetic hypothesis for 22 species representing five genera of the subtribe Coelogyninae.As proposed by De Vogel , the genl et al.  proposedPholidota in traditional circumscription is highly polyphyletic, consisting of at least four clades  comprising the generic type (P. imbricata) and representing the well-defined section Pholidota was well supported, and proved to cluster tightly together with its close relative P. pallida. The sister relationship between the Pholidota s.str. clade and a small clade composed by P. articulata (section Articulatae) and P. chinensis (section Chinenses) was also robustly recovered. As traditionally circumscribed, species of Pholidota are characterized by having a deeply saccate and boat-shaped lip hypochile [Coelogyne even for the amateur. Despite striking differences in their floral features, however, the Pholidota s.str. subclade appeared as paraphyletic relative to subclade II that includes two representatives of Coelogyne . This result is in agreement with previous studies inferred from plastid fragments and ITS sequences where a similar relationship was weakly supported [Coelogyne members for further analyses.The remaining des Fig. . Within ypochile , 2. Thisupported . As the Panisea had uncertain position in previous studies [Panisea, P. uniflora and P. protracta of Pholidota section Crinonia. Besides, a small clade comprising P. longipes and P. niana was also clustered into the subclade III  and two accessions of P. yunnanensis (sect. Repentes). These results indicate that neither section Repentes nor section Chinenses are monophyletic. Our data suggest a close relationship between Bulleyia and Pholidota species of these two sections. Species of Bulleyia is typically circumscribed based on a distinctive lip gradually narrowed at base into an incurved spur [In particular, we investigated the systematic position of the monotypic genus  IV Fig. , B. yunnved spur . In factved spur , the indPholidota and its related genera are far more complex than previously expected. Thus, additional studies using more samples and more molecular data are needed to clarify the phylogeny and evolutionary history of Pholidota and its allies before a formal taxonomic revision.Our results highlight the homoplasious character of lip base that usually used for circumscribing generic boundaries. As these characters might represent homoplasies resulting from similar pollination systems rather than synapomorphies, the discriminatory characters must be chosen carefully in Coelogyninae. Owing to ambiguous diagnostic characters and similar floral features, the phylogenetic and taxonomic relationships of those species traditionally accommodated in Pholidota were sequenced and assembled for the first time. These plastid genomes were found highly conservative in gene content, order and structure. Nonetheless, comparative analyses identified the divergence of the boundaries between LSC/SSC and IR regions. The multiple genetic markers, including repetitive sequences, SSRs, SNPs and InDels were proved to be highly efficient to evaluate the genetic diversity. Six hypervariable regions were selected that are potentially informative for barcoding, phylogenetic and population genetic researches. Our phylogenomic analyses yielded a well-resolved tree and identified highly supported novel phylogenetic relationships between Pholidota and its related genera. Our data provide strong support for the positions of Bulleyia and Panisea. Combined with the previous studies, we considered that the current taxonomy system of Pholidota needs to be improved and revised. The results further highlighted the advantage of plastome to infer phylogenies of putatively rapidly radiating groups. Moreover, the genomic data generated in the present study provide the basis for better understanding of the diversification of Pholidota and increasing the taxonomic resolution, species identification of Pholidota.In this study, the complete chloroplast genomes of thirteen species of Pholidota representing major evolutionary lineages were sampled. Total genomic DNA was isolated from the young leaf using a modified CTAB method [Fresh leaf materials were collected from healthy living plants cultivated at the greenhouses of the National Orchid Conservation & Research Center of Shenzhen (NOCC) and South China Botanical Garden, Chinese Academy of Science . In total, 13 accessions of B method . The purPholidota plastomes have been deposited in the NCBI GenBank database with the accession number ON880551–ON880563  [The relative synonymous codon usage (RSCU) of a codon represents the ratio of its observed frequency of utilization divided by the frequency expected if all the synonymous codons were used equally. An RSCU value equal to 1 denotes no bias in codon usage. RSCU value < 1.00 indicates a less-frequent usage, whereas RSCU value > 1.00 indicates a positive codon usage bias, as a codon is used more frequently than expected . To detei (1987) .https://bibiserv.cebitec.uni-bielefeld.de/reputer/) was utilized to analyze dispersed  and palindrome repeats in Pholidota plastomes. For identification of these oligonucleotide repeats, the following conditions were used: a minimum repeat size of 30 bp; a hamming distance equal to 3 . In addition, Tandem Repeats Finder v.4.09 [https://tandem.bu.edu/trf/trf.html) was employed to detect tandem repeats under default parameters. Simple sequence repeats (SSRs) or microsatellites in the genomes were examined using the Perl script-based program, MISA v.1.01 [REPuter v.2.74 program  (https:/r v.4.09  (https:/A v.1.01 . Differehttps://server.gview.ca/) with 100 bp connection windows [BLAST Atlas on the GView server  were included as outgroups based on previous study [http://iqtree.cibiv.univie.ac.at). The best-fitting nucleotide substitution model TVM + F + R3 was determined using the Akaike Information Criterion (AIC) by ModelFinder [To better clarify the evolutionary relationships within the subtribe Coelogyninae, phylogenetic analyses were carried out based on 22 complete cp. genomes. Except for the 13 newly generated us study . All theus study  and adjuus study . Phylogeus study  and web elFinder  in the IelFinder , based oelFinder . These tBelow is the link to the electronic supplementary material.Additional file 1: Figure S1.?A graphical circular map generated using the Gview server, showing a full view of the thirteen Pholidota plastomes with P. longipes as a reference. The innermost ring shows the genome size in kbp, followed by GC skew in purple, GC content in black, protein-coding genes on both the forward and reverse strand. The remaining rings display BLAST comparisons of plastome sequences. From the inside to the outside: P. ventricosa, P. missionariorum, P. cantonensis, P. leveilleana, P. wenshanica, P. yunnanensis, P. chinensis, P. articulata, P. pallida, P. imbricata, P. protracta, P. niana, P. longipes. The similar and divergent locations are shown in continuous and interrupted track lines, respectively. The lightly screened areas from the inside radically out denote divergent regions with high levels of variations.Additional file 2: Figure S2. Codon usage frequency based on relative synonymous codon usage (RSCU) values in the thirteen Pholidota cp genomes.Additional file 3: Table S1. Codon frequencies and relative synonymous codon usage (RSCU) values of the thirteen Pholidota cp genomes.Additional file 4: Table S2. Types and numbers of long repeats in the thirteen Pholidota cp genomes.Additional file 5: Table S3. Distribution of tandem repeats in the thirteen Pholidota cp genomes.Additional file 6: Table S4. Types and numbers of SSRs distributed in LSC, SSC, IR regions based on nucleotide repeat units in the thirteen Pholidota plastomes.Additional file 7: Table S5. The statistics of SNPs and InDels identified for each plastome alignment with Pholidota cp genome as a reference.


Staphylococcus aureus bacteremia (SAB) is one of the most common and clinically significant bacterial infections worldwide. Malnutrition has been linked to poor outcomes in various infectious and non-infectious diseases. The objective of this study is to investigate the impact of malnutrition on the outcomes among adult patients hospitalized with SAB.Staphylococcus aureus bacteremia were identified and stratified by the presence of malnutrition using ICD-10 codes. The primary outcome was in-hospital mortality. Secondary outcomes included ICU admissions, septic shock, mechanical ventilation, length of stay (LOS), and hospital charges. Multivariate regression analysis adjusted for confounding factors.A cohort study was conducted using the Nationwide Inpatient Sample (2016-2019). Adult hospitalizations with Methicillin-Sensitive or -Resistant Of 518,840 SAB patients, 14.10% had malnutrition. Malnourished patients showed higher rates of mortality, septic shock, mechanical ventilation, mean LOS, and hospital charges. Multivariate analysis revealed malnutrition as an independent predictor of worse outcomes. Adjusted odds ratios were 1.60 for mortality (P< 0.001), 1.69 for septic shock (P< 0.001), and 1.75 for mechanical ventilation (P< 0.001). Adjusted mean LOS and total charges increased by 6.14 days and $87,885 for malnourished SAB patients. Outcomes were similar for MSSA and MRSA.Our study highlights the significant impact of malnutrition on outcome among patients hospitalized with SAB. We found that malnourished individuals with SAB had higher rates of mortality, septic shock, mechanical ventilation, prolonged hospital stays and increased financial burden. These results emphasize the critical need to address and recognize malnutrition as a significant risk factor in the management of SAB. Further research and interventions targeting the issue of malnutrition in SAB patients are needed to enhance outcomes, decrease associated mortality and morbidity, and reduce healthcare cost.All Authors: No reported disclosures


Paenibacillus polymyxa complex have broad-spectrum activities against fungi and bacteria. Their antibacterial activities against soft rot Dickeya and Pectobacterium phytopathogens containing multiple polymyxin-resistant genes were not clear. Here, we selected nine strains within the P. polymyxa complex having broad-spectrum antagonistic activities against phytopathogenic fungi and a polymyxin-resistant D. dadantii strain causing stem and root rot disease of sweet potato and did antagonistic assays on nutrient agar and sweet potato tuber slices. These strains within the P. polymyxa complex showed clear antagonistic activities against D. dadantii in vitro and in vivo. The most effective antagonistic strain P. polymyxa ShX301 showed broad-spectrum antagonistic activities against all the test Dickeya and Pectobacterium strains, completely eliminated D. dadantii from sweet potato seed tubers, and promoted the growth of sweet potato seedlings. Cell-free culture filtrate of P. polymyxa ShX301 inhibited D. dadantii growth, swimming motility, and biofilm formation and disrupted D. dadantii plasma membranes, releasing nucleic acids and proteins. Multiple lipopeptides produced by P. polymyxa ShX301 may play a major role in the bactericidal and bacteriostatic actions. This study clarifies that the antimicrobial spectrum of polymyxin-producing bacteria within the P. polymyxa complex includes the polymyxin-resistant Dickeya and Pectobacterium phytopathogens and strengthens the fact that bacteria within the P. polymyxa complex have high probability of being effective biocontrol agents and plant growth promoters.Polymyxin-producing bacteria within the  Pectobacterium and Dickeya bacteria produce multiple pectinases and cause destructive soft rot diseases of numerous crops and ornamental plants and great economic losses worldwide  × 100. The in vivo inhibition rate (%) was calculated by [1-(diameter of maceration zone by D. dadantii with Paenibacillus/diameter of maceration zone by D. dadantii without Paenibacillus] × 100. strains  using thP. polymyxa stain ShX301 was determined using the in vivo assay with sweet potato seed tubers as previously described + and [M+K]+. Fusaricidins also appeared as open-chain products [M+H+H2O]+ [m/z 802–865, m/z 904–946, m/z 984–1052, m/z 1101, m/z 1122, m/z 1144–1225, m/z 1542–1559 and m/z 1753) in the range of m/z 800–1800 from CFCS of P. polymyxa ShX301  to resist polymyxins [Pectobacterium carotovorum (formerly Erwinia carotovora) [D. dadantii CZ1501 resists 2 μg·mL−1 of polymyxin B, multiple polymyxins produced by P. polymyxa ShX301 may accumulate to a locally high concentration, contributing to the cytotoxic action on the damage of D. dadantii plasma membrane and the inhibition of D. dadantii growth in vitro, in vivo, and in planta. Pelgipeptins produced by Paenibacillus have broad-spectrum activities against Gram-positive and Gram-negative bacteria and fungi, including phytopathogens [Bacillus and have potent antifungal activity and limited antibacterial activity [P. carotovorum [P. polymyxa ShX301 may also contribute to the cytotoxic action on D. dadantii.MALDI-TOF-MS revealed that bacteria ,31 and tbacteria , while Dlymyxins . Howeverotovora) . Here, aathogens ,29,33,34activity , have alotovorum . The pelD. dadantii to colonize plant surface, intercellular spaces, and xylem vessels and complete disease cycles [P. polymyxa ShX301 inhibited D. dadantii swimming motility and biofilm formation. Fusaricidins and polymyxins from P. polymyxa may not play a major role in cytotoxic action on D. dadantii but may act as biosurfactants to inhibit surface attachment and biofilm formation [D. dadantii infection and increasing D. dadantii susceptibility to cytotoxic lipopeptides. Multiple lipopeptides produced by P. polymyxa ShX301 play a major role in bactericidal and bacteriostatic actions on the phytopathogens.Flagella-mediated motility and biofilm formation facilitate e cycles ,37,38,39ormation , thus reP. polymyxa [P. polymyxa ShX301 completely eliminated D. dadantii from sweet potato seed tubers at 21 d after the equivalent inoculation of the two bacteria. Bactericidal and bacteriostatic actions and induced plant systemic resistance mediated by multiple lipopeptides may play together to eliminate D. dadantii. The latent infection of seed tubers by Dickeya and Pectobacterium is a major source of the soft rot diseases of potato and sweet potato plants [P. polymyxa ShX301 has shown the potential to control soft rot diseases from seed tuber-borne pathogens. Moreover, inoculating P. polymyxa ShX301 alone to sweet potato seed tubers promoted the growth of sweet potato seedlings. Likewise, P. polymyxa ShX301 promoted the growth of cotton seedlings and suppressed the soil-borne fungal pathogen Verticillium dahlia and the Verticillium wilt disease of cotton seedlings [P. polymyxa ShX301 is a promising biocontrol agent and plant growth promoter for future application in fields and study of the underlying mechanisms of the broad-spectrum antagonism and mutually beneficial interactions with plants.From the view of plant-microbe interactions, plants can induce the expression of polymyxin and fusaricidin biosynthesis genes in polymyxa , while lpolymyxa ,43. Notao plants . P. polyeedlings . TherefoP. polymyxa complex includes the polymyxin-resistant soft rot Dickeya and Pectobacterium pathogens and strengthens the fact that bacteria within the P. polymyxa complex have high probability of being effective biocontrol agents and plant growth promoters. P. polymyxa ShX301 will be used to clarify the biocontrol mechanism of the P. polymyxa complex against broad-spectrum phytopathogens, including polymyxin-resistant Dickeya and Pectobacterium and to control plant diseases in fields.This study clarifies that the antimicrobial spectrum of polymyxin-producing bacteria within the


Ureaplasma urealyticum  infection is primarily associated with damage to male fertility through its effects on male sperm parameters. However, its effects on sperm semiological variables remain unclear. Therefore, this study aimed to investigate whether U. urealyticum infection was associated with semen quality and sperm morphology.U. urealyticum infection were studied using general linear models.From 2019 to 2021, this cross-sectional study analyzed infective pathogens and semen variables in 1064 males (22–30 years old) recruited from our reproductive center and the general public. Routine semen parameters and normal sperm morphology rate were analyzed using methods outlined by the World Health Organization. The associations between semen quality, sperm morphology, and U. urealyticum infection (n=328), (ii) non-U. urealyticum infection , and (iii) normal volunteers . U. urealyticum in semen samples was observed to be associated with lower sperm concentrations (p<0.001) and a lower ratio of anterograde motile spermatozoa (p<0.001). Semen cultures positive for U. urealyticum were associated with lower normal sperm morphology (p<0.001) compared to semen cultures negative for U. urealyticum.The participants were categorized into three groups: (i) U. urealyticum during routine clinical examinations and diagnoses of males with infertility.This study shows the importance of proper investigations for  Inflammatory reactions and factors caused by U. urealyticum infection likely affect the surrounding environment and normal sperm function. Moreover, U. urealyticum infection is considered a common infection that contributes to changes in the internal structure of sperm cells. Studies have reported that current clinical mycoplasma infections in the urogenital system of infertile males are mainly caused by U. urealyticum , Neisseria gonorrhea (NG), molds, and trichinosis might also cause genital tract infections  evaluated by sperm chromatin structure assay (SCSA)  .U. urealyticum infection in homogeneous males with infertility.Therefore, due to the controversies in current research, we aimed to study the incidence and impact of male genital 22.1U. urealyticum, compared with semen parameters, as a pathogen in urogenital tract infection.Data for this study were collected from a randomized controlled trial that investigated the association of U. urealyticum infection and male fertility were approved by the ethics Review Committee of Anhui Province Maternal and Child Health Hospital. Furthermore, this study was conducted in accordance with the Declaration of Helsinki.This study adopted the definition of male infertility by the World Health Organization , which is the inability of a male to fertilize his partner after >12 months of cohabitation with normal sexual intercourse and no contraceptive measures . Based oU. urealyticum infection, including males with U. urealyticum detected in their semen samples, n=328), (ii) non-U. urealyticum infection, including males with urogenital tract infection symptoms but no U. urealyticum detected in their semen samples, n=377), and (iii) normal volunteers, including males with no urogenital tract infection symptoms and no pathogens detected in their semen samples, n=359).Data collected from the 1064 males were as follows (1): duration of the abstinence period before semen sample delivery (2), semen routine parameters and normal sperm morphology rate, and (3) pathogens in the semen. Based on the results of laboratory cultures, the participants were divided into three groups: (i) 2.2Samples of human semen were collected by masturbation during an abstinence period, which was standardized to be 3–5 days in line with the WHO 2010 guidelines . The exa2.3Routine semen analysis was performed according to the WHO Laboratory Manual for the Examination and Processing of Human Semen (5th edition) . Semen v6/ml) was obtained using an automatic semen quality computer-aided analysis system . Other data collected were the rates of forward sperm (%), non-forward sperm (%), inactive sperm (%), survival rate (%), average path velocity (um/s), linear motility (%), wobble (%), linear (%), and straightness (%), as well as curve velocity (um/s), the amplitude of lateral head display (um), beat cross frequency (Hz), mean angular displacement, and straight-line velocity (um/s).Semen volume (mL), viscosity, liquefaction degree, and appearance were measured manually according to standard operating procedure (SOP) specifications, and the pH was tested using a litmus test paper. Next, 10 µL of each semen sample was drawn using a sampling gun on a sperm counting plate . The sperm concentration  to analyze the ratio of normal sperm morphology . Briefly2.4U. urealyticum. U. urealyticum was detected using polymerase chain reaction (PCR) with specific kits . A standard curve was formulated for each test based on the kit contents. Concentrations of U. urealyticum DNA ≥500 copies/ml were considered positive  were calculated. Statistically significant differences were considered at p-values <0.05, and SPSS 20.0 was used for all statistical analyses.Patients’ age, semen volume, pH value, liquefaction time, sperm concentration, forward moving sperm, non-forward moving sperm, viability, average path velocity, curve velocity, the amplitude of lateral head display, beat cross frequency, mean angular displacement, straight-line velocity, wobble, linear, straightness, as well as the rates of normal morphology, head deformity, and middle and main segment deformity, and round cell number, were measured and distributed normally, expressed as mean ± standard deviation (x ± s). One-way ANOVA was used to compare the differences between groups. Viscosity was a classified variable, and non-parametric tests were used for statistical analyses. 33.1U. urealyticum. Approximately 377 (35.43%) males were considered to have symptoms of urogenital infections based on their responses to the questionnaires; however, U. urealyticum was not detected in their semen samples. In contrast, 359 (33.74%) males were observed to have no urogenital infection symptoms, and U. urealyticum was not detected in their semen samples.3.2U. urealyticum infection group than in the non-U. urealyticum infection and normal volunteers groups  (Semen pH and liquefaction time were significantly higher in the ctively) Table 3.3.3U. urealyticum infection was significantly associated with lower ratios of sperm with normal morphology , compared with semen males without U. urealyticum infection . This result was higher than that in most studies .After bination , and U. movement . Infectimovement . The effU. urealyticum infection in semen samples significantly increases the rate of sperm malformation . The detection and treatment of U. urealyticum are recommended for males with infertility to improve their semen quality.This study observed that male genital tract infection with The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Each author contributed to the search process. The experiments were conceived and designed by FX, FJ, DZ. Carry out experiments: FX, FJ, DZ. HM. Analysis data: FX. Compilation manual: FJ. The manual has been strictly revised, and the final version has been completed/corrected: FX, FJ, HM. All authors contributed to the article and approved the submitted version.


Plant-parasitic nematodes (PPNs) that cause most damage include root-knot nematodes (RKNs) which are a major impediment to crop production. Root-knot nematodes, like other parasites, secrete proteins which are required for parasite proliferation and survival within the host during the infection process.Meloidogyne javanica genome. Furthermore, functional annotation analysis was performed using various integrated bioinformatic tools to determine the biological significance of the predicted secretome. In total, 7,458 proteins were identified as secreted ones. A large percentage of this secretome is comprised of small proteins of ≤ 300 aa sequence length. Functional analyses showed that M. javanica secretome comprises cell wall degrading enzymes for facilitating nematode invasion, and migration by disintegrating the complex plant cell wall components. In addition, peptidases and peptidase inhibitors are an important category of M. javanica secretome involved in compatible host-nematode interactions.Here, we used various computational tools to predict and identify classically and non-classically secreted proteins encoded in the M. javanica genome. Future experimental validation analyses can greatly benefit from this global analysis of M. javanica secretome. Equally, our analyses will advance knowledge of the interaction between plants and nematodes.This study identifies the putative secretome encoded in the The online version contains supplementary material available at 10.1186/s12864-023-09366-6. Meloidogyne  are some of the most yield-limiting plant-parasitic nematodes (PPNs). On a global scale, PPNs cause huge economic losses in the agricultural sector with annual losses estimated at $US157 billion MeloidogyM. graminicola identified putative secretory proteins [M. incognita secretory proteins in a stage-specific manner [M. incognita genome [M. incognita [Global transcriptomic analysis of pre-parasitic and parasitic phases of proteins . Moreovec manner , 15. Seca genome . In addincognita .M. incognita using in silico tools, proteomics, and genome-wide approaches. This could be due to the availability and accessibility of M. incognita genome sequences earlier than other RKN species [Meloidogyne spp. including M. arenaria, M. javanica, M. hapla, M. graminicola, M. floridensis, M. luci, M. exigua, and M. enterolobii [Meloidogyne spp.In view of the above, it is evident that previous studies have largely focused on identification of secretory proteins from  species , its hig species . Now, wierolobii –24 genomM. javanica genome sequences allowed us to explore its secretome systematically and comprehensively using bioinformatics approaches. Indeed, computational tools have been used widely in the discovery of putative ESPs in PPNs and animal parasitic nematodes (APNs) [M. javanica secretome. The present study not only adds to the secretory proteins repertoire from M. javanica but also provides a resourceful biological database that lays a framework for future functional characterization of these putative M. javanica secretory proteins. In addition, this research provides a foundation for understanding the interaction between RKNs and their host plants.The availability of s (APNs) , 25–27. M. javanica secreted proteins. Of the 97,208 proteins encoded in the M. javanica genome, 5,258 and 2,369 were identified as classically and non-classically secreted proteins, using SignalP and SecretomeP softwares. The 7,627 secreted proteins identified were without transmembrane domains according to TMHMM and Phobius analysis. TargetP analysis identified 11 proteins as mitochondrial-target proteins, and these were discarded.Nematode secretions released by the infective juveniles are crucial for a successful infection process and participate in various stages of nematode parasitism . Here, whttps://www.ncbi.nlm.nih.gov/nuccore/?term=Meloidogyne+javanica). This implies that these proteins are expressed during the nematode infection cycle and may be involved in nematode parasitism.Further, PS-SCAN was used to scan endoplasmic reticulum-based proteins while PreGPI detected secretory proteins but linked to the membrane by a GPI anchor. In total, 26 proteins with endoplasmic reticulum retention signal and 132 proteins predicted to be GPI anchored proteins were removed from the data set. Thus, the bioinformatic pipeline predicted a total of 7,458 (7.7%) of the entire proteome as the secretome, and these were used for further analysis Fig. . OverallThe size distribution analysis of the predicted secreted proteins showed that 50.1%  of the proteins were between 101 and 300 aa, followed by proteins with a sequence length of below 100 aa accounted for 32.4% . The remaining secretome is made up of 906 (12.1%) proteins with sequence lengths between 301 and 500 aa, 344 (4.6%) proteins between 501 and 1000 aa, and 56 (0.2%) proteins with more than 1000 aa  identified in the promoter of Meloidogyne incognita dorsal gland encoding effectors [M. javanica secretome. The core motif was detected in the 2 kb upstream of the coding region of 1,608 secretory proteins out of the 7,256 promoter regions retrieved from WormBase database  were assigned to 3,513 GO terms with 1,044 GO terms from biological process category, 1,216 GO terms from molecular function and 1,253 GO terms from cellular component category. A summary of the GO term annotation is shown in Fig. GO terms such as cellular , and metabolic process GO:0008152; 47.2%), biological regulation , regulation of biological process , developmental process  and response to stimulus  were the most represented GO terms under biological process category  binding , and molecular function regulator  , membrane part (48.9%) cell , cell part  organelle , and extracellular region  were found to be highly represented GO terms within this category  I63 (90 proteins), I35 (19 proteins), and I43 (25 proteins) among others , C-type lectin-like domain, Transthyretin-like, CAP (cysteine-rich secretory proteins) domain, Pancreatic trypsin inhibitor Kunitz domain, Modulator of levamisole receptor-1, Peptidase S10 serine carboxypeptidase, Pectate lyase PlyH/PlyE-like, Glycoside hydrolase, family 5, and DnaJ domain.Using InterProScan domain search, 1915 (25.7%) of the predicted secreted proteins were mapped into 412 known protein domains and families. The most abundant InterProScan domains summarized in Table Out of the 7,458 predicted secretory proteins, 672 (9.0%) were mapped into 470 KEGG pathways presented in Additional file 2: Table M. javanica were searched for homologues in other closely related Meloidogyne species. From this comparative analysis, the tropical Meloidogyne spp. shared 323 orthologous groups with 1,737 proteins which can be explored further to uncover typical mechanisms RKNs use to undermine host immunity and promote disease processes. A set of 599 clusters with 1,684 proteins was specific to M. javanica secretome  protein whose function is yet to be known.We subjected the 382 protein sequences to a fold recognition analysis using the Phyre2 algorithm to obtain additional functional information for this set of specific secreted proteins. The Phyre2 tool determines the structure and function of novel protein sequences using advanced homology detection algorithms , 35. In M.javanica secreted proteins with homologues in C. elegans. M. javanica proteins homologous to C.elegans RNAi lethal phenotype genes can be potential targets for functional analysis using RNA interference (RNAi) tool and inform designing of control strategies. Meloidogyne javanica protein sequences with homologues in host proteomes were excluded from this analysis considering that they are less likely to be exploited as nematode control targets due to off-targets effects. In total, 5,289 secretory proteins were detected as RKN-specific shown in Additional file 1: Figures C. elegans’ lethal phenotype genes. On the premise that homologous genes retain similar functions in other organisms and share other crucial features [C. elegans RNAi lethal phenotype genes including transthyretin-like protein family, peptidases, and peptidase inhibitors . This work represents the first in silico prediction of M. javanica secretome which accounts for 7.7% of the total proteome. The size of M. javanica secretome is comparable to those reported in other PPNs in comparison to their proteome size [The ever-increasing number of sequenced genomes of PPNs provides an unparalleled opportunity to delve into their secretome, which is critical for their ability to survive within host cells. To this end, we applied various computational approaches to predict and annotate the secretome encoded in the ome size , 27, 41 M. javanica secretome, reported in this study with the secretome of M. incognita predicted by [M. javanica and M. incognita secretome, given that Meloidogyne species have the same lifestyle, as obligate biotrophs [ that are probably involved in virulence and mediate parasite-host interactions [We examine the composition of RKNs secretomes by comparing the functional annotation of icted by . Paralleiotrophs . One obshs [Fig.  that areractions , 44.. These molecular activities are associated with the functions of CWDEs. RKN predicted proteins were mapped into various CWDEs families, namely glycoside hydrolase, glycosyltransferase, polysaccharide lyases, and carbohydrate-binding modules. CWDEs provide an essential catalog of RKN secretomes, which not only facilitate nematode’s penetration and migration through host tissue, but they also help nematodes utilize plant nutrients as a source of food and develop nematode feeding sites [Molecular entities linked to hydrolase activity, lyase activity, and carbohydrate-binding activity under MF were enriched in the secretome compared to the proteome in the GO enrichment analysis Fig. . These mng sites , 45, 46.ng sites . As a reM. javanica infection process [M. graminicola pectate lyase (Mg-PEL1) revealed that it is involved in virulence and facilitates RKN parasitism [Pectate lyases and glycoside hydrolase 5 represent the most abundant families found in this study and are also among the differentially expressed effector-encoding genes implicated in cell wall modification during  process . The funrasitism .M. javanica secretome harbors secretory proteins with distinct protein domains which are largely shared with the closely related M. incognita secretome. The protein kinase domain  was, for example, one of the most represented domains according to InterProscan analyses  and thioredoxin (IPR013766) domains  which were supported by our domain analysis (Table ). Both catalytic classes play important roles in PPN infection processes such as host tissue invasion, nematode development, reproduction, and pathogenicity. Furthermore, it has been demonstrated that silencing cysteine and serine peptidases in parasitic nematodes is detrimental to nematode fecundity and parasitism [Enzyme regulator function was found enriched in mes Fig.  which aroidogyne . The mosne [Fig.  which weis Table . Both carasitism –59. Peptrasitism , 60. TheM. javanica and M. incognita secretomes, it was noteworthy that secondary and pigment metabolic processes, developmental processes, and anatomical developmental structure were enriched in M. javanica secretome  but not in M. incognita secretome [Although there are parallels between ome Fig.  but not ecretome . Secondaecretome . Though ecretome .Meloidogyne species secretomes was carried out to uncover species-specific M. javanica secretome. The species-specific proteins may reveal the parasite-specific molecular mechanism of host adaptation and specialization [M. javanica-specific secreted proteins were discovered that may help to explain nematode lifestyle, virulence, and host specificity and could eventually be applied to design control strategies for M. javanica infestations.Further comparative analysis with different lization . Here, 3M. javanica parasitic survival, as shown by functional groups identified such as defense and cell death. Remarkably, positive regulation of siRNA production and regulation of miRNA-mediated gene silencing were among the represent GO terms. Current research indicates that pathogen small RNAs play an active role in virulence [These secreted proteins might be critical in mediating irulence . HoweverM. javanica. To the best of our knowledge, this is the first account of M. javanica secretome being identified and functionally annotated. In silico analysis of secretomes encoded by parasites’ genomes can precede and complement the laborious and complex experimental methodologies applied in deciphering the function of putative excreted-secreted proteins.In conclusion, this study sheds light on what constitutes the secretome of M. javanica genome under BioProject ID PRJEB8714 [M. javanica 7,734 expressed sequence tags (EST) retrieved from the NCBI dbEST database (https://www.ncbi.nlm.nih.gov/genbank/dbest/) using tblastn at a cut-off value of 1e-05 and percentage identity of ≥ 50%.The proteome deduced from RJEB8714  was retrRJEB8714  was applRJEB8714 . Both seRJEB8714  and PhobRJEB8714  for the RJEB8714  with a pRJEB8714   associated with the expression of dorsal gland effectors [All the secretory proteins were scanned for the presence of the recently identified core motif Mel-DOG  terms, GO enrichment analysis, mapping protein domains, and pathway associations using KEGG pathways. The secretory proteins were assigned to Gene Ontology (GO) terms into three functional categories of Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) using the PANNZER2 functional annotation web server . GO visu37138727 . Signifi37138727  using BL37138727 . Protein37138727 .The KAAS   was usedMeloidogyne species including M. incognita, M. enterolobii, and M. floridensis we performed orthologous gene clusters using orthovenn2 with the following parameters; e-value 1e-05 and an inflation value 1.5 [To identify putative orthologs in the secretome of closely related alue 1.5 . The speM. javanica secretome was searched for similarity against the host proteomes  using Orthovenn 2 default parameters; e-value 1e-05 and an inflation value 1.5 [Caenorhabditis elegans lethal RNA interference (RNAi) phenotypes genes retrieved from the WormMine database (WS284) using the tblastn program and filtered using an e-value threshold of 1e-05 and a percentage identity of 50%.The predicted alue 1.5 . The resBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2