HMO alone did not affect overall microbial composition, but increased (p < 0.05) the relative proportion of specific taxa, including Blautia, compared to other groups. Bacteroides abundance was increased (p < 0.05) in the AC by BMOS and synergistically by BMOS + HMO in the feces. Distinct effects of HMO and BMOS suggest complementary and sometimes synergistic benefits of supplementing a complex mixture of OS to formula.Milk oligosaccharides (OS) shape microbiome structure and function, but their relative abundances differ between species. Herein, the impact of the human milk oligosaccharides (HMO) (2′-fucosyllactose [2′FL] and lacto-N-neotetraose [LNnT]) and OS isolated from bovine milk (BMOS) on microbiota composition and volatile fatty acid (VFA) concentrations in ascending colon (AC) contents and feces was assessed. Intact male piglets received diets either containing 6.5 g/L BMOS (n = 12), 1.0 g/L 2′FL + 0.5 g/L LNnT , both , or neither  from postnatal day (PND) 2 to 34. Microbiota were assessed by 16S rRNA gene sequencing and real-time PCR, and VFA were measured by gas chromatography. The microbiota was affected by OS in an intestine region-specific manner. BMOS reduced ( Breastfeeding is the recommended form of feeding human infants and is associated with both short- and long-term health benefits ,2. ExcluWhile commercial infant formula have undergone significant modifications over the past few decades with the additional of bioactive ingredients, such as prebiotics, lactoferrin, and milk fat globule membrane , dissimiBifidobacterium, while suppressing potential pathogens [Several studies have reported beneficial effects of feeding HMO and bovine milk derived oligosaccharides (BMOS), including modulation of growth , alkalinathogens ,25,26,27athogens ,27.To date, two HMO, 2′-FL and LNnT, are commercially available and have been added to infant formula ,29; up tDue to the high degree of similarity in anatomy, physiology, immunology, and brain development patterns between pigs and humans, piglets are considered an ideal model for neonatal nutrition research ,33,34. PThe study design and diets composition were previously described by Fleming et al. . BrieflyBMOS was generated from whey permeate of bovine milk containing galactooligosaccharides (GOS) and other OS from bovine milk, such as 3′- and 6′-sialyllactose . The OS w/v ratio) in Eppendorf tubes and stored at −20 °C until analyses were performed.Ascending colon (AC) contents and feces were collected on PND 34. AC contents were collected as most dietary OS are fermented in the colon. Fecal samples were also studied as the microbiota composition of colon contents differed from that of feces  and studDry matter (DM), pH, and VFA  concentrations were measured on AC contents and feces. Dry matter was assessed on the samples according to the AOAC method . pH was DNA was isolated from AC contents and feces by combination of the QIAamp Fast DNA Stool Mini Kit  with bead beating on the FastPrep-24 System , as previously described . The congg_13_8_otus/taxonomy/97_otu_taxonomy.txt, accessed on 24 February 2017) [Sequences were demultiplexed at the sequencing facility with the bcl2fastq v2.17.1.14 Conversion Software , allowed 0 mismatches in the barcode sequences. De-multiplexed forward and reverse reads were processed using the QIIME pipeline (version 1.9.1) . Brieflyry 2017) . An OTU Bacteroides fragilis, Clostridium perfringens, Clostridium difficile, and Escherichia coli [Bifidobacterium spp. and Lactobacillus spp., as members of these genera utilize milk OS [Prevotella spp. contains enzymes involved in mucin oligosaccharide degradation [Prevotella abundance was associated with high fiber intake [B. fragilis and E. coli) or SYBR Green  assays as previously described [8 16S rRNA gene copies/reaction). Data analysis was processed with QuantStudio software V1.3 supplied by Thermo Fisher Scientific  and presented as 16S rRNA gene copy numbers/g of AC contents or feces.Real-time PCR was used as a complementary approach to sequencing in order to study the effect of dietary treatment on opportunistic pathogenic species that are associated with infection in infants, including Enterobacteriaceae, hia coli ,46,47, a milk OS  and provradation  and an ir intake . Real-tiescribed  using prp < 0.05.All data were analyzed as 2 × 2 factorial design. Differences in overall bacterial community structure among dietary treatments were evaluated with distanced-based redundancy analysis (db-RDA). db-RDA were performed on unweighted UniFrac distances using capscale command of Vegan package of R . The stap = 0.0025 and p = 0.0136, respectively). HMO supplementation had no effect on AC or fecal DM and pH . The addition of BMOS to the diets increased the acetate concentrations in AC regardless of the presence of HMO (p = 0.0473). Fecal acetate concentration was highest in HMO + BMOS and CON, intermediate in BMOS and lowest in HMO piglets, while butyrate concentrations were highest in HMO + BMOS, intermediate in CON and BMOS and lowest in HMO (p < 0.05). Neither HMO nor BMOS affected propionate, isobutyrate, valerate or isovalerate concentrations at either sampling location.The percent dry matter was lower in piglets fed either BMOS containing diets (BMOS and HMO + BMOS groups) than in piglets consumed diets without BMOS (CON and HMO groups) in both AC contents and feces  across the 85 samples. After assembling of paired-end reads, quality filtering and removing of singletons, 3,970,157 sequences  were utilized for further analysis.Pmodel = 0.190); however, a significant effect of BMOS was observed (p = 0.012) when BMOS was considered as the only main effect (p = 0.0432) and Tenericutes (p = 0.0032) were lower in the AC of piglets consuming diets with BMOS (BMOS and HMO + BMOS groups) than without BMOS (CON and HMO groups). At the genus level, BMOS supplementation decreased relative abundances of Dorea, Eubacterium, Peptococcus, Lactococcus, Desulfovibrio, and unclassified Mogibacteriaceae, whereas it increased unclassified Preovotellaceae in the AC (p < 0.05).Distance-based redundancy analysis based on unweighted Unifrac distances indicated that the addition of BMOS (BMOS and BMOS + HMO groups) did not affect the overall microbiota composition in the AC contents when both BMOS and HMO were included in the statistical model (n effect . Differep = 0.0148), Chao1 (p = 0.0321) and Faith PD (p = 0.0066) were lower in AC of piglets fed diets with BMOS (BMOS and HMO + BMOS groups) than diets without BMOS (CON and HMO groups). A similar effect on Chao1 was also observed in AC of piglets fed HMO-containing diets (HMO and HMO + BMOS) (p = 0.0275). In feces, observed OTUs. Chao1 and Faith PD did not differ among the diet groups (p > 0.05); however, Simpson reciprocal index was highest in BMOS, intermediate in HMO and HMO + BMOS, and lowest in CON group (p < 0.05). Neither HMO nor BMOS supplementation affected Shannon index (p > 0.05).The effects of BMOS and HMO on alpha diversity are summarized in Prevotella spp. in AC and Enterobacteriaceae and E. coli in feces were higher in piglets fed diets containing BMOS  than diets without BMOS . HMO had no effect on abundances of any bacterial groups/species measured by RT PCR (p > 0.05)  .Colonization of gut microbiota plays an essential role in host metabolism, development of neonatal gastrointestinal, immune, and neural systems, and also affects short- and long-term health outcomes ,54. The Although bovine milk contains lower concentration of OS than human milk, the two share at least 10 common structures ,61. BothPrevotella spp. in AC contents, but not in feces. Prevotella spp. is one of the three major genera that differentiated individual gut microbiota into enterotypes [Prevotella were associated with long-term high fiber intake in humans [In this study, high-throughput sequencing was applied to assess the effect of BMOS on the global profile of gut microbial communities. We demonstrated BMOS altered overall bacterial community structures and modulated the relative abundances of bacterial taxa in both AC contents and feces. Effects of BMOS on microbiota differed at the 2 intestinal sampling sites. For instance, BMOS increased the abundances of erotypes . Previoun humans . In our n humans  and diffn humans . Furthern humans .Similarly, BMOS-containing formula elicited significant effects on VFA concentrations. The VFA are bacterial fermentation end-products of dietary fibers and amino acids and their profiles represent the collective metabolic activity of the gut microbiota. OS are minimally digested by hydrolytic/enzymatic digestion in the upper gastrointestinal tract and most dietary OS reach the colon where they are subsequently fermented by the gut microbiota . In our Bifidobacterium to a level closer to breastfed infants [The HMO, 2′FL and LNnT, are highly abundant in human milk. 2′FL is the predominant oligosaccharide in the milk of secretor mothers, representing nearly 30% of total HMO with a mean level of 2–3 g/L . The con infants .Bifidobacterium were low in piglets and there were no difference among the dietary groups. These contradictory observations likely arise from the fundamental differences in the gut microbiota composition of infants and piglets. The predominant phyla of piglets are Bacteroidetes and Firmicutes [Bifidobacterium, is generally dominant in infant stool microbiota regardless of how they are fed. While Bifidobacterium is detectable in the gastrointestinal tract of piglets, the relative abundance is considerably lower (<0.1% of 16S rRNA gene sequences) than in infants. Furthermore, the Bifidobacterium species detected in piglets differed from that of human infants. B. longum subsp. infantis, B. longum subsp. longum, B. breve, B. catenulatum, and B. adolescentis represent the predominant species of human infants, while Bifidobacterium found in piglets are B. longum subsp. suis, B. globosum and B. pseudolongum [Bifidobacterium species and strains [Bifidobacterium species, and hardly detected in the genomes of Bifidobacterium species isolated from human adults and animals [B. longum subsp. infantis [In contrast with the results of human infant study, neither Faith PD nor overall bacterial structure were impacted by HMO supplementation in the piglets in this study. Furthermore, relative abundances of rmicutes ,67,68, wrmicutes . Actinobdolongum ,71. A re strains . Additio animals . We haveinfantis , thus fuinfantis .Blautia were higher in the AC contents of piglets supplemented with HMO alone compared to other groups. Blautia is a genus in the bacterial family Lachnospiraceae that phylogenetically belongs to Clostridium Cluster XIVa within phylum Firmicutes [Blautia is widely distributed among fecal samples of human and animals [Blautia are efficient degraders of dietary fibers and producers of short chain fatty acids [Blautia may play a role in human health outcomes. Compared to healthy children, the abundance of Blautia was significantly lower in the fecal microbiota of children with autism spectrum disorders, but higher in children with type 1 diabetes [Blautia spp. in humans during the neonatal period, warranting further studies.The relative abundances of rmicutes ,75. Blau animals ,76. Membdiabetes ,78. DespBacteroides is a predominant genus found in the gut of human adults, while its abundance varies greatly in infant feces with some infants being dominated by Bacteroides rather than Bifidobacterium [Bacteroides are effective consumers of milk OS [Bifidobacterium and Bacteroides in breastfed infants [Bacteroides was the predominant bacterial genus in both AC contents and feces of piglets; whereas the relative abundances of Bifidobacterium are very low (<0.1% of sequences). This is perhaps not a surprising result as genomic analysis of Bacteroides spp. revealed a specialized machinery encoded by the polysaccharide utilization loci (PULs) dedicated to the import and processing of HMO, plant polysaccharides and mucins [Bifidobacterium species isolated from pig, including B. globosum and B. pseudolongum, lack HMO-utilizing genes [Bifidobacterium spp. cannot utilize milk OS, supplementation of HMO + BMOS provides a selective advantage for Bacteroides growth in the gut of piglets. Given human milk contains both OS and bacteria and that microbiota of vaginally-delivered breastfed infants is typically predominated by Bifidobacterium [Bifidobacterium that avidly consumes OS, especially for infants whose early microbiota are dominated by other bacteria, such as Bacteroides and Enterobacteriaceae, and not with HMO-utilizing Bifidobacterium [Human milk contains both neutral and acidic OS. 2′FL and LNnT are neutral OS, while BMOS are rich in acidic OS such as 3′SL and 6′SL. The biological activities of HMO are dependent upon their chemical structures, we hypothesized that the combination of HMO and BMOS would more closely mimic the structural complexity and biological functions of OS present in the human milk. We observed synergistic effects on gut microbiota composition when HMO + BMOS were supplemented to formula. The relative abundance of fecal Bacteroides was 33% higher in piglets receiving BMOS + HMO than piglets consuming BMOS (19%) or HMO (17%) alone. acterium . In vitr milk OS ,80 and a infants ,80. In tng genes . Therefoacterium ,82, our Blautia. Synergistic effects, such as an increase in Bacteroides, were observed when combination of HMO and BMOS were added to formula. Taken together with the observed OS-specific effects on recognition memory, both absolute and relative volumes of cortical and subcortical brain regions, and hippocampal mRNA expression in these same piglets [Supplementation of BMOS and BMOS + HMO modulated microbiota composition and VFA profiles in the neonatal model investigated herein. The addition of HMO did not affect overall bacterial communities; however, HMO alone increased the proportion of specific taxa, such as  piglets  demonstr


Amaranthus dubius is a leafy vegetable widely cultivated in Asia and Africa. The complete chloroplast genome of Amaranthus dubius was sequenced and assembled in this study. The complete chloroplast genome is 150,520 bp. A total of 130 genes were identified, including 85 protein-coding genes, eight rRNA genes, and 37 tRNA genes. The overall GC content of this genome was 36.6%. The phylogenetic tree based on 10 chloroplast genomes in Amaranthaceae supports that A. dubius is sister to A. hypochondriacus and A. caudatus. Amaranthus dubius Mart. ex Thell. 1912 (Amaranthaceae), an annual herb native to tropical America, is widely cultivated as a green vegetable and considered a medicinal herb in many Asian and African countries . DNA was extracted from its dried specimen leaves using Plant Genomic DNA Kit . The specimen and extracted DNA was deposited at Shanghai Chenshan Herbarium (CSH), Shanghai Chenshan Botanical Garden (http://www.csnbgsh.cn) under the voucher number RQHD03140 (collected by Xiao-Ling Yan: sx_yxl@163.com). The plastome sequences were generated using the Illumina HiSeq 2500 platform . In total, about 16 million high-quality clean reads (150 bp PE read length) were generated with adaptors trimmed. Aligning, assembly, and annotation were conducted by GetOrganelle  is 150,520 bp. It is comprised by a large single copy region , a small single copy region , and two inverted repeat regions . The GC content of A. dubius chloroplast genome was 36.6% and the GC contents of the LSC, SSC, and IR regions are 34.5%, 30.3%, and 42.6%. The genome  contained 130 genes. Seventeen genes had two copies, which were comprised of six PCG genes , seven tRNA genes , and all four rRNA species . In the genome, seven protein-coding genes  had one intron, and three protein-coding genes  contained two introns.The full length of A. dubius in Amaranthus, chloroplast genomes sequences of additional eight Amaranthus species and Alternanthera philoxeroides as outgroup were obtained from NCBI. The sequence alignment was conducted by MAFFT v7.450 (Katoh and Standley A. dubius is sister to a clade formed by A. hypochondriacus and A. caudatus (A. dubius will provide necessary genetic resource and background data for further phylogenetic study of the Amaranthaceae.To analyze the phylogenetic placement of caudatus . We hope


Mycobacterium avium complex (MAC) pulmonary disease six years before admission. Etanercept was discontinued two years ago because of MAC pulmonary disease progression and restarted nine months before admission because of worsening arthritis, again resulting in MAC pulmonary disease progression. Etanercept was discontinued again; however, the pulmonary disease progressed more rapidly. The condition was considered paradoxical worsening caused by IRIS due to etanercept discontinuation. The disease resolved quickly with chemotherapy for MAC.Antitumor necrosis factor–associated nontuberculous mycobacteria-immune reconstitution inflammatory syndrome (IRIS) has rarely been reported. An 84-year-old woman with a history of rheumatoid arthritis treated with etanercept was diagnosed with  The use of anti-TNF therapy has increased the incidence of anti-TNF–associated tuberculosis . It has The incidence of NTM diseases has recently been increasing worldwide . Further2Mycobacterium avium complex (MAC) pulmonary disease on the basis of a new lesion observed on her chest radiograph and sputum culture results, and it was observed because she remained asymptomatic and her MAC lesion was stable for some time. However, the pulmonary lesion expanded gradually, and etanercept was discontinued two years before admission due to onset of respiratory symptoms. Nine months before admission, etanercept was restarted owing to worsening arthritis; however, it was discontinued seven months before admission because of exacerbation of respiratory symptoms, such as cough and sputum. Even after etanercept discontinuation, home oxygen therapy for respiratory failure was started five months before admission. Chest computed tomography (CT) findings showed slight expansion of the MAC lesions in the lungs. Methotrexate was discontinued; however, the patient's clinical course continued to worsen. Finally, she visited the Chubu Hospital due to worsening hemoptysis and dyspnea.The patient was an 84-year-old Japanese woman with a 42-year history of rheumatoid arthritis. She had started receiving methotrexate therapy 12 years before admission; etanercept, a TNF inhibitor, was added to the therapy ten years before admission. Six years before admission, she was diagnosed with M. intracellulare/chimaera using mass spectrometry. Chemotherapy for MAC  was initiated, resulting in an improvement in her respiratory symptoms within two weeks. Furthermore, her radiological findings promptly improved. Chemotherapy for MAC was discontinued six months later due to the appearance of refractory drug rashes. The MAC pulmonary disease has remained stable for over two years after chemotherapy.The clinical course and chest radiographs are shown in Seven months before admission, the patient experienced clinical deterioration, even after etanercept discontinuation. Chest radiographs show lesion expansion in the right upper and lower lung fields. Additionally, the patient experienced chronic respiratory failure and required oxygen therapy. Finally, she was admitted because of worsening hemoptysis and respiratory failure. All clinical manifestations rapidly resolved after the initiation of chemotherapy.33.1IRIS is a well-known complication observed in human immunodeficiency virus (HIV)-infected patients undergoing antiretroviral therapy and is defined in these patients as worsening of inflammatory symptoms during immune reconstitution due to antiretroviral therapy initiation . RegardiTNF inhibitors are considered to increase the risk of NTM disease ; however3.2M. marinum infection in the joints. Etanercept and methotrexate were discontinued, and chemotherapy was initiated; however, synovitis progressed within four months. Infliximab was eventually initiated to control inflammation. Subsequently, the treatment was successfully completed [M. intracellulare pulmonary disease 16 months after the latest adalimumab administration. Tacrolimus was discontinued, and chemotherapy for MAC was initiated; however, the clinical condition worsened, and the patient developed disseminated MAC infection. Steroid dosage was increased, and treatment was subsequently completed [The cases of anti-TNF–associated tuberculosis-IRIS have often been reported , whereasompleted . In the ompleted . Althougompleted . Some ofompleted . The thiPatients with anti-TNF–associated tuberculosis-IRIS sometimes treated successfully with the administration of corticosteroids and/or the re-administration of TNF inhibitors as anti-inflammatory agents . The rev4•Paradoxical worsening of NTM pulmonary disease in the presented patient was considered IRIS due to etanercept discontinuation•Although anti-TNF–associated NTM-IRIS is rare, clinicians should consider the possibility of IRIS when NTM diseases worsen in patients receiving anti-TNF therapyThis report did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.There are no ethical issues regarding this manuscript. The medical record of the patient was retrospectively reviewed and identifying information removed.Conceptualization - D.Nabeya.Data curation - D.Nabeya.Formal analysis - D.Nabeya.Funding acquisition - None.Investigation - D.Nabeya.Methodology - D.Nabeya.Project administration – T.Kishaba.Resources – D.Nabeya, T.Kishaba.Software – None.Supervision – T.Kishaba.Validation – D.Nabeya, T.Kishaba.Visualization - D.Nabeya.Roles/Writing - original draft - D.Nabeya.Writing - review & editing – D.Nabeya, T.Kishaba.None.


Mycobacterium abscessus is a rapid growing, free-living species of bacterium that also causes lung infections in humans. Human infections are usually acquired from the environment; however, dominant circulating clones (DCCs) have emerged recently in both M. abscessus subsp. massiliense and subsp. abscessus that appear to be transmitted among humans and are now globally distributed. These recently emerged clones are potentially informative about the ecological and evolutionary mechanisms of pathogen emergence and host adaptation. The geographical distribution of DCCs has been reported, but the genomic processes underlying their transition from environmental bacterium to human pathogen are not well characterized. To address this knowledge gap, we delineated the structure of M. abscessus subspecies abscessus and massiliense using genomic data from 200 clinical isolates of M. abscessus from seven geographical regions. We identified differences in overall patterns of lateral gene transfer (LGT) and barriers to LGT between subspecies and between environmental and host-adapted bacteria. We further characterized genome reorganization that accompanied bacterial host adaptation, inferring selection pressures acting at both genic and intergenic loci. We found that both subspecies encode an expansive pangenome with many genes at rare frequencies. Recombination appears more frequent in M. abscessus subsp. massiliense than in subsp. abscessus, consistent with prior reports. We found evidence suggesting that phage are exchanged between subspecies, despite genetic barriers evident elsewhere throughout the genome. Patterns of LGT differed according to niche, with less LGT observed among host-adapted DCCs versus environmental bacteria. We also found evidence suggesting that DCCs are under distinct selection pressures at both genic and intergenic sites. Our results indicate that host adaptation of M. abscessus was accompanied by major changes in genome evolution, including shifts in the apparent frequency of LGT and impacts of selection. Differences were evident among the DCCs as well, which varied in the degree of gene content remodelling, suggesting they were placed differently along the evolutionary trajectory toward host adaptation. These results provide insight into the evolutionary forces that reshape bacterial genomes as they emerge into the pathogenic niche. Previously published sequencing data used in this study are available from the National Center for Biotechnology Information (NCBI) under the Sequence Read Archive (SRA) accession numbers found in Table S1 (available with the online version of this article).Mycobacterium abscessus is an important pathogen that causes skin abscesses, and pulmonary and other infections, and is a common bacterial pathogen isolated from cystic fibrosis patients. Until recently, M. abscessus was thought to be acquired exclusively from the environment; however, new evidence of person-to-person transmission has serious implications for how we monitor and treat these infections, particularly with antibiotic-resistant strains. Our study leverages powerful genomics tools to characterize the recent emergence of M. abscessus into the human pathogenic niche. We find significant genomic changes associated with strains originating from person-to-person transmission including changes in patterns of lateral gene transfer, and evidence for relaxed purifying selection. Our work highlights the importance of genome remodelling in the process of pathogen emergence.Mycobacterium abscessus is a rapid growing mycobacterial species found in a variety of environments [M. abscessus is the most common species of non-tuberculous mycobacterium (NTM) isolated from individuals with cystic fibrosis (CF) [M. abscessus infections are hard to treat, as the bacteria are intrinsically resistant to many antibiotics and additional resistances are on the rise [M. abscessus was thought to be exclusively acquired from the environment; however, evidence of person-to-person transmission has been identified for recently emerged dominant circulating clones (DCCs) [ronments . This frronments . M. absis (CF) . M. abthe rise . Until rs (DCCs) . DCCs ars (DCCs) .M. abscessus is distantly related to Mycobacterium tuberculosis and other pathogenic mycobacteria. M. abscessus has been divided into three subspecies: abscessus, massiliense and bolletii [M. abscessus are consistent as described in other species: within subspecies ANI values are >98 %, while between subspecies ANI values are ~96–97 % [M. abscessus subsp. abscessus recombines more frequently than the other subspecies [M. abscessus encodes a diverse array of plasmids and prophages [M. abscessus is unknown.bolletii . Average~96–97 % . It was bspecies . M. abrophages ; the extM. abscessus provides an opportunity to learn about the mechanisms underlying bacterial pathogen emergence, including the role of LGT. LGT can enable bacterial adaptation to new environments, including the pathogenic niche. Emergence of the major pathogen M. tuberculosis was accompanied by acquisition of novel genetic material by LGT [M. abscessus. We found putatively host-adapted DCCs to be characterized by lower levels of LGT relative to environmentally acquired M. abscessus. We did not observe large-scale pseudogenization across the DCCs; however, we found evidence of relaxation of purifying selection in the core and accessory genomes of DCCs, which may be an earlier indicator of host adaptation. Taken together, these results provide insight into genome reorganization that accompanies bacterial pathogen emergence.The recent emergence of host-adapted lineages of l by LGT , followel by LGT . Previoul by LGT . Our aimet al. [M. abscessus reference ATCC 19977 genome. Our pipeline can be found at https://github.com/pepperell-lab/RGAPepPipe (accessed 20 July 2021). Briefly, we used FastQC v0.11.8 [M. abscessus ATCC 19977 genome using SAMtools v1.5 [http://broadinstitute.github.io/picard/). Lastly, we called variants using Pilon v1.16 [M. abscessus isolates by subsp. abscessus and massiliense, and used FastTree v2.1.9 [M. abscessus subsp. abscessus DCC1 and DCC2, and M. abscessus subsp. massiliense DCC1, following the nomenclature described by Bryant et al. [M. abscessus subsp. abscessus and 80 subsp. massiliense isolates. We included isolates from subspecies abscessus and massiliense. We did not include isolates from M. abscessus subspecies bolletii, because there were few isolates available and there have not been reports of host-associated DCC emergences within the subspecies [We compiled Illumina sequence read data from a collection of 554 clinical isolates including newly sequenced isolates from Norway (PRJEB9515), as well as publicly available isolates from Michigan PRJNA315990), Maryland (PRJNA231221), and the sample published by Bryant et al. . We mapp v0.11.8  and Trim v0.11.8  for qualols v1.5 , and use5990, Mare v2.1.9  to recone v2.1.9  to identt et al.  to inforet al. [Bacterial isolates received at the Norwegian Institute of Public Health were grown on Lowenstein–Jensen media. Genomic DNA was extracted as described in Eldholm et al. . SequencM. abscessus genomes were de novo assembled with SPAdes v. 3.12.0 [de novo assemblies using Quast v. 4.6.3 [prank [The . 3.12.0  and anno. 3.12.0 . We assev. 4.6.3  and remov. 4.6.3  with a a3 [prank . AdditioWe performed maximum-likelihood phylogenetic inference on core-genome alignments using RAxML v 8.2.3  with theM. abscessus subspecies to inferred recombinant tracts in other mycobacterial species known to participate in distributive conjugal transfer (DCT): 'Mycobacterium canettii' [Mycobacterium smegmatis [Recombinant tracts were identified in the core genomes using Gubbins v 2.3.2  and visuanettii'  and Mygmatis . We usedgmatis  to recongmatis .M. abscessus subspecies. We constructed a core biallelic SNP alignment across all 200 M. abscessus isolates using SNP-sites [We performed bacterial admixture analyses using FineSTRUCTURE v. 2.1.3  to invesNP-sites  and VCFtNP-sites . Next, wNP-sites . AdditioM. abscessus subsp. abscessus and subsp. massiliense isolates, as well as DCCs and EAIs for both subspecies using random subsampling without replacement 100 times, plotting the median value for the core and pangenome values. Additionally, we calculated the gene frequencies of accessory genes from both subspecies. Using EggLib [We calculated pangenome accumulation and rarefaction curves of g EggLib , we calcg EggLib . We theng EggLib . To inveg EggLib .https://cran.r-project.org/web/packages/adephylo/index.html) package in R. For core distances, the separate core-genome phylogenies for each subspecies were used . For accessory distances, we used the phylogenetic tree based on accessory gene presence/absence as output by Roary .Core- and accessory-genome tree distances were calculated for all pairs of isolates within a subspecies using the distTips function from the adephylo  was measured for random subsamples of EAIs and DCC isolates . From each subsample, the core-genome and pangenome sizes  were plotted against the number of segregating sites calculated using EggLib [R2 values and P values were plotted using the stat_poly_eq and stat_fit_glance methods from ggpmisc (https://cran.r-project.org/web/packages/ggpmisc/index.html).In order to measure core-genome divergence for samples, as opposed to pairs of isolates, we calculated segregating sites for the separate core-genome alignments of g EggLib . A lineak-mer (sequences of length k) content between prophage nucleotide sequences. To visualize the relatedness between prophage regions, we plotted a dendrogram in R.We used ProphET  to detecblast [M. abscessus proteins and blasted the IGRs against the custom database. Pseudogenes were identified if the blast result had an E value cut-off of 10e−5 as described by Belda and colleagues [To identify and characterize possible pseudogenes, we looked for identity between intergenic sequences in our alignment and known protein sequences. Specifically, we constructed an alignment of all IGRs using Piggy  with a mblast  databaselleagues . We perflleagues . We thenlleagues .M. abscessus subsp. abscessus and subsp. massiliense, we estimated dI/dS across intergenic alignments of M. abscessus subsp. abscessus DCC1, DCC2 and EAI, and M. abscessus subsp. massiliense DCC and EAIs, as described previously [paml [To identify the extent to which selection is acting on IGRs in the DCCs of both eviously . Brieflyeviously  in paml ly [paml . We calcde novo assembled and analysed core-genome alignments of 200 clinical M. abscessus (120 subsp. abscessus and 80 subsp. massiliense) isolates from seven geographical locations: Australia, Denmark, Dublin, Holland, Norway, the UK and the USA. Pangenome analysis of these 200 M. abscessus isolates defined a large accessory genome in both M. abscessus subsp. abscessus and subsp. massiliense, with many genes found at low frequencies. The core genome consisted of 3672 genes present in at least 99 % of isolates, whereas a total of 41 939 genes were found at lower frequencies (Table S2). The mean number of genes per isolate was comparable between M. abscessus subsp. abscessus and subsp. massiliense: 5182 and 5107, respectively.We M. abscessus subsp. abscessus and subsp. massiliense to be clearly differentiated, as shown by the long branch separating them in the maximum-likelihood phylogeny and network , suggesting M. abscessus subsp. massiliense participates in LGT more than M. abscessus subsp. abscessus. Both subspecies did, however, show evidence of recombination (PHI (pairwise homoplasy index) test of recombination, P=0). Pangenomes of the two subspecies are of similar size . Using Gubbins [M. abscessus subsp. massiliense has been affected by recombination, which compares to 91 % for M. abscessus subsp. abscessus   . We foune in DCT    .M. abscessus subsp. abscessus and subsp. massiliense, we found several lines of evidence suggesting there are barriers to accessory gene flow between subspecies. First, shared accessory genes are maintained at different frequencies between the two subspecies  . These results suggest accessory genes are transferred more frequently within than between subspecies.When comparing the accessory genomes of bspecies . In the M. abscessus subsp. massiliense or subsp. abscessus  , suggesting that the M. abscessus pangenome does not expand linearly as a sample diversifies. There was a significant correlation between core-genome size and sample diversification for both subspecies   . In the or both) ; that isR2 0.24 for M. abscessus subsp. massiliense, 0.21 for subsp. abscessus; P<0.01 for both) suggesting that accessory-genome differentiation occurs in parallel with differentiation of the core genome. Synthesizing this observation with findings at the sample level, we infer that rates of acquisition of novel genes parallel rates of core-genome diversification, but that the pool of novel genes for EAIs is very large and unstructured such that the overall size of the pangenome does not expand linearly as a sample diversifies. We also analysed EAI–DCC pairs  and found that for a given core-genome distance, accessory gene content of DCCs is more differentiated from EAIs than EAIs are from each other. This is consistent with genetic isolation of DCCs. Taken together, these results suggest that the pangenome dynamics of the DCCs are distinct from those of the EAIs, likely due to the DCCs losing access to the large pool of accessory genes shared among EAIs.We also examined the relationship between core- and accessory-genome diversification at the level of individual isolates . Here, we compared tree distances from the core-genome phylogeny with accessory-genome distances inferred from a gene presence/absence matrix for pairs of EAIs. Examining pairs of isolates in this way, we observed a linear relationship between core- and accessory-genome distances for EAIs from both subspecies  were intermingled . This suggests that, relative to the M. abscessus subsp. massiliense DCCs, the M. abscessus subsp. abscessus DCC may be further along an adaptive trajectory away from the EAIs and toward host association and specialization.In addition to differences in recombination, we identified other genomic differences between DCCs and EAIs. blast method described previously [The genomes of bacterial pathogens are often observed to be smaller relative to their free-living relatives and it has been posited that ‘genome downsizing’ is a feature of host adaptation . Pseudogeviously . The datM. abscessus subsp. abscessus DCC1 vs EAI: Mann–Whitney–Wilcoxon test, W=2.9e5, P=0.0003) . This suggests DCCs could be evolving under relaxed purifying selection relative to EAIs.In order to investigate potential differences in selection pressures acting on DCCs and EAIs, we compared the distributions of pairwise πN/πS values across their core and accessory genomes. Gene-wise πN/πS values were higher in DCCs than in EAIs, for both subspecies .We hypothesized that the IGRs of the DCCs may be under distinct selection pressures relative to EAIs. To investigate this hypothesis, we calculated pairwise dI/dS and dN/dS values within and between groups for each subspecies. Distributions of dN/dS values were consistently lower than those of dI/dS, consistent with genic regions being under relatively strong selective constraint . Distribliense . This isDivergence time has been shown to affect overall patterns of genetic diversity, with a greater proportion of variation at non-synonymous sites observed among recently diverged bacteria . We hypoM. abscessus is a rapid growing, free-living mycobacterium that is capable of causing serious infections, particularly in people with CF and other lung diseases, as well as immunocompromised individuals [M. abscessus has increased substantially over the past decade [M. abscessus infections is challenging due to both intrinsic and acquired resistance to antimicrobials [ividuals . The inct decade . Treatmecrobials . The adacrobials .M. abscessus  loci ESX-1 and ESX-4 [M. abscessus encodes T7SS loci ESX-4 or ESX-4-bis/EVOL and ESX-3, as well as plasmids containing T7SS loci [M. abscessus engages in DCT, as we found recombinant fragments in the core genome to be similar in length to those of M. canettii, which is known to engage in DCT  [M. abscessus.DCT is another mechanism of LGT that has so far been uniquely identified in mycobacteria . Geneticnd ESX-4 . M. ab7SS loci . Our anaFig. S6) . Long reFig. S6) . Taken tM. abscessus subsp. massiliense engages in LGT more frequently than does subsp. abscessus [M. abscessus subsp. massiliense than subsp. abscessus . A previous report of whole-genome sequence data from three isolates mapped to different references interpreted mapping patterns as indicative of admixture between subspecies of M. abscessus and hypothesized that this was the result of DCT between subspecies [M. abscessus subsp. massiliense and subsp. abscessus to be genetically distinct, without evidence of substantial admixture M. absc with CF . A recencessus  found thcessus . This ecclonally . M. tueservoir . Similars suis . Free-liangenome , which wM. tuberculosis [M. abscessus and that this would be reflected in elevated dI/dS. DCCs were indeed characterized by relatively high rates of variation at intergenic sites . A recently published study  reportedet al. found that unlike symbionts, bacterial pathogen genomes were not characterized by excess pseudogenes or a loss of metabolic coding capacity.As noted above, pseudogenization has previously been identified in association with the shift to a host-associated lifestyle. We did not observe large-scale pseudogenization across the DCCs . This isM. abscessus populations are characterized by a complex structure, within which subspecies maintain genetic differentiation in their core and accessory genomes despite overall high levels of LGT mediated by multiple mechanisms. Phage are an exception in that they appear to be readily exchanged among subspecies, suggesting that barriers between subspecies are not maintained by specific pairings of host and phage. Evolving niche specialists, the DCCs, show evidence of genome remodelling and a dramatic shift in their mode of evolution with less evidence of LGT. This could reflect a loss of opportunity to partner with diverse bacteria and/or loss of the capacity to perform this function. The core genomes of DCCs exhibit varying levels of core-genome remodelling, consistent with successive emergence and slightly different placement along an evolutionary trajectory toward host adaptation. The DCCs appear to be under relaxed purifying selection relative to their free-living relatives, as has been observed in other pathogenic bacterial species. Consistent with recent research contrasting pathogens with other host-associated bacteria, we did not identify large-scale pseudogenization among the DCCs. Taken together, these results provide insight into adaptation of this important pathogen and the genomic remodelling events that occur during emergence into a new niche.In conclusion, Click here for additional data file.


Agriocnemis femina  and Ischnura senegalensis  are two damselflies inhabiting paddy lands. As an intermediate predator, they play an important role in controlling certain crop pest and mosquitoes. In this study, we sequenced complete mitogenomes of these two species. The total length of mitogenomes is 15,936 bp in A. femina and 15,762 bp in I. senegalensis. Both of mitogenomes consist of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and one control region. The close relationship between I. senegalensis and I. elegans was further proved by phylogenetic analysis. Our phylogenetic analysis indicated a clear two lineages in Coenagrionidae (Core and ridge-faced Coenagrionidae). Ridge-faced Coenagrionidae consisted of Megaloprepus caerulatus and Ceriagrion fallax. In core Coenagrionidae, Ischnura and Enallagma are most closely related; they formed one clade with Agriocnemis and then grouped together with Paracerion. Our study provides new genetic information for further study in phylogenetic analysis of Coenagrionidae. Agriocnemis femina, Brauer, 1868 and Ischnura senegalensis, Rambur, 1842). Both of these two species inhabit paddy land and become important predators for biological control of crop pests or mosquitoes  and I. senegalensis  were collected by sweep net in the Shenshan park of Wuhu, China  in July, 2019 and preserved in 90%-ethanol at room temperature. The specimen was deposited at Entomological Evolution and Ecology Room in Anhui Normal University  under the voucher number L502-SS03. Genomics DNA was extracted from muscle tissues of thorax using CTAB methods (Bechly http://mitos.bioinf.uni-leipzig.de) to annotate genomes  and 15,762 bp in I. senegalensis (Accession number in Genbank: MT787567). We noticed that a partial mitogenome of A. femina  is reported earlier. Comparing MK951677, 20 SNPs were found; one T-deletion in the interspace between trnT and trnP and one A-deletion and two A-insertions in 16S RNA coding region in MT787566 were found. Like other mitogenomes in Coenagrionidae, both new mitogenomes from us contain 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and one control region. All tRNA sequences length range from 63 to 72 bp. In both mitogenomes, the shortest tRNA sequence is trnG and the longest one is trnK. According to the predicted tRNA structure from MITOs, all tRNA sequences can form the characteristic clover leaf secondary structures. In both mitogenomes, trnS1 lacks DHU-stem and trnF lacks TΨC loop. For protein-coding genes, in A. femina eight genes  use ATG/ATA (encoding for methionine) and 5 genes  use ATT (encoding for isoleucine) as start codon; in I. senegalensis, nad6 use ATA and nad3 use ATT as start codon and the other genes are the same as in A. femina. We identified a standard stop codon for most of protein-coding genes; however, cox3 and nad5 in A. femina and nad5 in I. senegalensis had an incomplete stop codon.The total length of the new mitogenomes in our study is 15,936 bp in A. femina and I. senegalensis in Coenagrionidae, we collected 22 other mitogenome sequences from GenBank in Zygoptera (Lin et al. Anax imperator (Herzog et al. Brachythemis contaminata (Yu et al. I. senegalensis has a close phylogenetic relationship with I. elegans and forms one clade with I. pumilio. The close relationship between I. elegans and I. senegalensis can also be proved by their interspecific hybridization in the lab (Okude et al. Ischnura species formed one clade with Enallagma species and then clustered with newly sequenced A. femina. Therefore, the complete mitogenomes of I. senegalensis and A. femina in Coenagrionidae provide valuable genetic information for further phylogenetic analyses in Zygoptera.In order to determine the phylogenetic relationship of


Cinnamomum zeylanicum, Cinnamomum tamala, Amomum subulatum, Trigonella foenumgraecum, Mentha piperita, Coriandrum sativum, Lactuca sativa, and Brassica oleraceae var. italica. The antifungal potential was assessed via the disc diffusion method and, subsequently, the extracts were assessed for phytochemicals and total antioxidant activity. Potent polyphenols were detected using high-performance liquid chromatography (HPLC) and antifungal mechanism of action was evaluated in silico. Cinnamomum zeylanicum exhibited antifungal activity against all the tested strains while all plant extracts showed antifungal activity against Fusarium solani. Rutin, kaempferol, and quercetin were identified as common polyphenols. In silico studies showed that rutin displayed the greatest affinity with binding pocket of fungal 14-alpha demethylase and nucleoside diphosphokinase with the binding affinity , as compared to terbinafine. Results indicated that Cinnamomum zeylanicum and Cinnamomum tamala exert their antifungal effect possibly due to kaempferol and rutin, respectively, or possibly by inhibition of nucleoside diphosphokinase (NDK) and 14-alpha demethylase (CYP51), while Amomum subulatum and Trigonella foenum graecum might exhibit antifungal potential due to quercetin. Overall, the study demonstrates that plant-derived products have a high potential to control fungal infections.A worldwide increase in the incidence of fungal infections, emergence of new fungal strains, and antifungal resistance to commercially available antibiotics indicate the need to investigate new treatment options for fungal diseases. Therefore, the interest in exploring the antifungal activity of medicinal plants has now been increased to discover phyto-therapeutics in replacement to conventional antifungal drugs. The study was conducted to explore and identify the mechanism of action of antifungal agents of edible plants, including  Candida, Aspergillus, Fusarium species (spp.), and Mucor spp. [Fusarium spp., produces mycotoxins that can cause mycotoxicosis in humans upon ingestion of food, having a colony of this fungus. Fusarium solani induces skin lesions and onychomycosis [Aspergillus spp. mediated aspergillosis is increasing among patients undergoing chemotherapy and in patients with low immunity [Mucor spp. mediated mucormycosis is also widely reported [Aspergillus flavus [Fungal infections are continuously on the rise and one of the major causes of morbidity and mortality, particularly in immune-compromised patients ,2. Amongomycosis . Aspergiimmunity . Moreovereported . For thereported , all offreported ,8. Despis flavus . Moreoves flavus . Hence, Candida albicans and Saccharomyces cerevisiae [Candida auris by modifying fungal cell wall [To protect themselves against pathogens, plants produce and exude a myriad of secondary metabolites, which play important roles as defense mechanisms against various infections. The approach of traditional medicinal plant therapy is preferable due to fewer side effects and better efficacy . The userevisiae . Elagic ell wall . Anotherell wall . Nowadayell wall . In thisell wall . LikewisCinnamomum zeylanicum (C. zeylanicum), Cinnamomum tamala , Amomum subulatum (A. subulatum), Trigonella foenumgraecum (T. foenumgraecum), Mentha piperita (M. piperita), Coriandrum sativum (C. sativum), Lactuca sativa (L. sativa), and Brassica oleraceae var. italica (B. oleraceae)) and evaluated their antifungal potential. These common plants were selected due to their minimal/nontoxicity, significant antioxidant and antimicrobial properties, and their frequent use in routine diet. Active phytochemicals of plants were quantified using high-performance liquid chromatography (HPLC). Furthermore, in silico studies were performed to comprehend the underlying antifungal mechanism of detected polyphenols.Hence, assessment of the medicinal plants for antifungal potential should be prioritized, and the mechanism of action should be focused to discover novel phyto-therapeutic agents. In this contribution, we selected eight plants, including C. zeylanicum has the highest percent yield, while M. piperita presented a high percent recovery among herbs.Percent extract yield for selected herbs and spices was calculated, as shown in Fusarium solani. C. zeylanicum) inhibited the growth of all fungal strains with a maximum zone of inhibition (13.6 mm) against Fusarium solani. A. subulatum exhibited antifungal activity against Fusarium solani, Aspergillus niger, and Aspergillus flavus. Overall, extracts of T. foenumgraecum, C. zeylanicum, C. tamala, and A. subulatum showed greater antifungal activity against Fusarium solani than herbal extracts, i.e., C. sativum, L. sativa, B. oleraceae, and M. piperita (7.6 to 10 mm), as shown in Aspergillus flavus except C. zeylanicum and A. subulatum. M. piperita and T. foenumgraecum exhibited antifungal activity against Fusarium solani and Aspergillus niger while B. oleraceae possessed antifungal potential against Fusarium solani and Mucor spp. C. sativum and L. sativa exhibited antifungal potential only against Fusarium solani . Among ethanol extract of herbs, M. piperita showed the highest TPC (136.2 ± 6.4 mg GAE/g Extract), as presented in C. tamala > M. piperita > C. zeylanicum > A. subulatum > L. sativa Lactuca sativa > T. foenumgraecum > B. oleraceae > C. sativum. Among spices, T. foenumgraecum represented the highest TFC while M. piperita showed the highest TFC among herbs  and caffeic acid (0.43 ± 0.04 µg/mg extract) were quantified in M. piperita. Quercetin was found to be highest in T. foenumgraecum followed by A. subulatum. The representative chromatograms are shown in Quantitative polyphenol detection was carried out using HPLC , by comparing chromatograms of samples with that of standards . A totald (dissociation constant) values of −9.4 and −8.9, respectively  and nucleoside diphosphokinase (NDK) with the lowest Kectively . The lowosine 90 a, while,osine 90 b. Bindinosine 90 . Bindingne D:147 d,e, whilne D:147 d.C. zeylanicum, C. tamala, A. subulatum, T. foenumgraecum, M. piperita, C. sativum, L. sativa, and B. oleraceae var.italica were prepared and screened for their antifungal activities, and polyphenols were identified in each plant. The polyphenols are strong antioxidants that have an important role in controlling microbial diseases. The respective polyphenols were further assessed for interaction with fungal proteins, via in silico analysis. Major findings of the current research are: (i) C. zeylanicum, C. tamala, and A. subulatum displayed significant antifungal activity among studied plants; (ii) rutin, kaempferol, and quercetin were identified as the most common antifungal polyphenols in selected plants; (iii) rutin displayed the greatest affinity with binding pocket of fungal 14-alpha demethylase (CYP51) and nucleoside diphosphokinase (NDK) with the lowest Kd values as compared to standard drug terbinafine; (iv) C. zeylanicum is predicted to possess antifungal activity due to kaempferol  A. subulatum and T. foenumgraecum antifungal effect are likely due to quercetin; and (vi): rutin was the major polyphenol found in C. tamala and all herbs, which might be responsible for antifungal activity by either competitive or allosteric inhibition of both studied fungal enzymes.Increasing incidence of drug-resistant fungi, the emergence of new fungal strains, and the toxicity profile of antifungal drugs have led to the use of medicinal plants as potential antifungal means. Further, understanding the mechanism by which plant antifungal agents, i.e., polyphenols, interact with crucial fungal proteins should be of prime importance in identifying the molecular targets . Therefo C. zeylanicum showed the highest percent yield in comparison to other plant extracts. Different plants exhibited different yields, perhaps due to differences in their phytochemical composition and their variable solubility [of C. zeylanicum, C. tamala, and A. subulatum exhibited significant antifungal activities against different studied strains. A recent report has documented the antifungal efficacy of C. zeylanicum bark powder, its water suspensions, and its essential oils against Fusarium oxysporum. [C. zeylanicum methanol, n-hexane, and aqueous extracts are also reported to exhibit inhibitory effects against Alternaria solani [C. zeylanicum and A. subulatum against Fusarium solani, which we observed in our study. We found that C. zeylanicum and A. subulatum ethanolic extracts only moderately inhibited the growth of both Aspergillus species. In a previous report, methanolic extract of A. subulatum exhibited a considerably higher zone of inhibition  against Aspergillus niger [T. foenumgraecum is reported to inhibit the growth of fungal strains and exhibited inhibition effects against Fusarium oxysporum and Fusarium. Oxysporum [foenumgraecum has shown inhibition potential against Fusarium solani and Aspergillus niger. Extract of M. piperita inhibited the growth of two fungal strains, i.e., Aspergillus niger and Fusarium solani. M. piperita extract has been already shown to cause inhibitory effects against radial fungal growth and production of aflatoxin by Aspergillus species [B. oleraceae var.italica in our study inhibited the growth of Fusarium solani and Mucor spp.As per results of the current study, lubility . Furtherysporum. . The C. a solani . To the us niger . This mixysporum . In our  species . MoreoveC. zeylanicum leaf and bark extracts might be due to the di- and monohydroxyl substitutions in the aromatic ring, which possess potent hydrogen donating abilities as described by Shimada et al. [C. tamala, C. zeylanicum, A. subulatum, and M. piperita were possibly observed due to their high phenolic content. The significant antioxidant potential of these plants might be the reason for their antifungal efficacy, as plant flavonoids are reported to inhibit biofilm formation by stimulating membrane disturbances, which reduces the fungal cell size and causes leakage of intracellular components [Next, our results revealed that plants having antifungal activities exhibited excellent antioxidant potential, evaluated by free radical scavenging assay, total antioxidant capacity, and total reducing power activity. The reducing power of a et al. . Plants a et al. . It indimponents . Phenolsmponents . Hence, mponents ,30.C. zeylanicum. In a previous study, HPLC chromatogram showed the presence of quercetin and kaempferol in cinnamon extract [C. zeylanicum and A. subulatum ethanol extracts, respectively. In addition, rutin might be involved in the antifungal action of all other test extracts. Rutin and quercetin are reported to enhance the antifungal activity of amphotericin B [To detect the common antifungal polyphenols from plants under study, we performed HPLC analysis. Kaempferol was detected in  extract . Polyphe extract . Kaempfeericin B . Quercetericin B . Many stericin B . Althougericin B ,36γ-phosphate between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP) [d values of rutin (−9.4) were much lower than that of terbinafine (−8.9) which clearly indicates high inhibition potential of rutin for fungal CYP51 as compared to terbinafine. It may be as terbinafine and rutin both inhibit fungal ergostrol biosynthesis pathway but both work by inhibiting different enzymes of same pathway. In another report, fluconazole and ketozole showed binding affinity values of −7.6 and −10, respectively, with fungal alpha demethylase [d values against fungal NDK, which is reported for regulation of the spore development and pathogenicity [To explore the underlying mechanisms inhibiting the fungal growth, in silico modeling was carried out which further confirmed the inhibition of fungal enzymes by the polyphenols detected in the extracts. We evaluated the inhibition potential of detected polyphenols against two fungal enzymes, i.e., CYP51 and NDK. The CYP51 belongs to the cytochrome P450 monooxygenase superfamily and mediates a crucial step of the synthesis of ergosterol, which is a fungal-specific sterol. The NDK catalyzes the reversible exchange of the te (NDP) ,38. Amonte (NDP) . In the te (NDP) . In our te (NDP) . As terbte (NDP) , in the ethylase ,43. It igenicity . Quercetgenicity . AccordiOther detected polyphenols such as vanillic acid, ferulic acid, catechin, and caffeic acid also have antifungal properties  but theyC. zeylanicum, C. tamala, A. subulatum, and T. foenumgraecum) and herbs  were collected during September 2020 from the local market of Bharakahu, Islamabad, Pakistan. The specimens were identified by Dr. Muhammad Zafar, Department of Plant Sciences, Quaid-i-Azam University, Islamabad.Spices , in air-tight glass bottles. Maceration was performed to increase the contact between plant material and solvent (Ethanol) and to soften plant’s cell wall so that plant phytochemicals soluble in ethanol may be released. Sonication (after maceration) was added to further enhance the disruption of the plant cell wall and facilitate the release of phytochemicals. The extracts were filtered using Whatman #1 filter paper . Extracts were concentrated by vacuum evaporation in a rotary evaporator  and dried in a vacuum hood at 40 °C, and stored at 15 °C till further use. The extracts were filtered using Whatman #1 filter paper. Extracts were concentrated by vacuum evaporation in a rotary evaporator  and dried in a vacuum hood at 40 °C, and stored at 15 °C till further use.The extracts, once dried, were weighed to find out % recovery by the following formula:v/v tween 20 in H2O) and the turbidity was adjusted according to McFarland 0.5 turbidity standard. Then, fungal strain (100 µL) was streaked on sabouraud dextrose agar. Discs of filter paper, impregnated with test extract , positive control , and negative control (DMSO) were employed on agar plates and incubated . Afterward, the average diameter (mm) of the growth inhibition zone around all the discs was measured and recorded.The antifungal activity of extracts was evaluated by the agar disc diffusion method, performed in triplicate . The spow/v in H2O). The mixture was incubated  and then 500 μL of trichloroacetic acid was added. Afterward, centrifuged at room temperature (10 min). The upper layer (500 μL) was mixed with eqal volumn of distilled water and FeCl3 . Then, 200 μL was takenfrom this was taken and the absorbance of the was measured at 645nm using microplate reader . The assay was performed in triplicate. The chemicals and standards used in the assays were purchased from Sigma-Aldrich .The reducing power assay was performed as described by Ahmed et al., with some modifications . Briefly2O) in 96 well plate. After incubation for 90 min at 95 °C in the water bath, the reaction mixture was cooled at room temperature and absorbance was taken at 630 nm using microplate reader . Both TRP and TAC were expressed as the number of mg of vitimin C (vit. C) equivalents per gram of extract (vit. C eq/g). The chemicals and standards used in the assay were purchased from Sigma-Aldrich.Total antioxidant capacity was determined by the phosphomolybdenum method . An aliqDiscoloration of purple-colored DPPH was used to measure the free radical scavenging activities of extracts ,46. Here50) was determined using Graph Pad Prism 5 software.The inhibitory concentration at which the test sample showed 50% scavenging  per gram of extract (mg GAE/g Extract). The standard gallic acid and other chemicals used in this assay were purchased from Sigma-Aldrich.TPC was measured by the Folin–Ciocalteu method as reported previously . Brieflyw/v in H2O), 1.0 M potassium acetate (10 μL), and distilled water (170 μL) were mixed and incubated at room temperature (30 min). The absorbance of the mixture was calculated at 405 nm.The TFC of the extracts was determined by aluminium chloride colorimetric assay, with modifications . Briefly2 value of 0.996 were obtained.The resultant TFC was expressed in microgram equivalents of quercetin per milligram Extract (µg QE/mg Extract). The standard curve equations for quercetin (Sigma-Aldrich) (y = 0.01x − 0.004) and the RPolyphenols were detected and quantified using HPLC analysis of medicinal plants. HPLC system, Agilent Chem station Rev. B.02–01-SR1 (260), equipped with a Zorbex-C8 analytical column  in combination with a diode array detector  was used, following previously reported method . All HPLMolecular docking was performed to predict the possible antifungal mechanisms of polyphenols, detected via HPLC analysis. Protein databank (PDB) was used to obtain 3D structures of fungal enzymes 14-alpha-demethylase and nucleoside diphosphokinase with PDB IDs 5FRB and 6K3H, respectively. A 3D structure CYP51B protein was modeled using Swiss Prot. Pubchem was used to obtain structures of ligands . Ligands and heteroatoms were removed and proteins were optimized and minimized using UCSF Chimera software to obtain structurally correct protein. Docking was performed using the Pyrx-virtual screening tool, binding affinities were saved as CSV files and Discovery studio was used to visualize the final protein–ligand interactions .Statistical analysis was carried out using SPSS statistics and MS Excel. Replication of each experiment was conducted thrice.C. zeylanicum, C. tamala, and A. subulatum represent good sources of such antifungal polyphenols. Bio-formulation involving extracts from more than one plant may be necessary for effective antifungal application. Our study suggested that high-phenolic content of plant extracts is responsible for their antioxidant and fungal-inhibitory activity. These tested plants may be used for developing new, safer, and effective fungicides. Moreover, detected polyphenols can be used either alone or synergistically with the fungal antibiotics to reduce their toxic effects and to increase the antifungal efficacy.This study revealed new scientific insights about the antifungal activity of natural polyphenols and their mechanism of action.


Candida spp are capable of infecting both normal and immunocompromised individuals. More recently, Candida infections have spread considerably in healthcare settings, especially in intensive care units, where it is the most frequently encountered pathogen. Candida albicans is the commonest species encountered, although infections by non-albicans species have also risen in the past few years. The pathogenicity of Candida is credited to its aptitude to change between yeast and hyphal modes of growth. Candida spp produce biofilms on synthetic materials that protect them and facilitate drug resistance and act as a source for chronic and recurrent infections. Primarily, azoles antifungal agents are utilized to treat Candida infection that targets the ergosterol synthesis pathway in the cell wall. The development of antifungal resistance in Candida species is a major reason for treatment failure, and hence, there is a need to develop newer antifungal molecules and/or modifications of existing antifungals to make them more effective and less toxic. This has led researchers to oversee the plants to discover newer antimicrobials. Middle Eastern countries are well known for their landscape ranging from dry and sandy deserts to snow-capped mountains. However, they comprise enormous plant diversity with over 20,000 different species showing various types of bioactivities, such as anticancer, antidiabetic, and antimicrobial activities. Especially, the antifungal potential of these phytoproducts could be exploited in the clinical setting for therapy. The present review examines some of the promising alternative natural compounds that have been tested and found effective in treating Candida infections in vitro in some Middle Eastern countries. Candida spp are ubiquitous and the most common human fungal pathogens that attack both immunosuppressed and immunocompetent individuals and is considered a major healthcare-related infection [Candida is mostly unicellular. Although more than 200 species of Candida have been identified, only a few species are infectious to humans. Candida albicans remains the main species that have been implicated in most of the Candida infections, although infections by nonalbicans species have also risen in the past few years [C. albicans could reproduce by budding as well as by hyphal form that fragments intermittently to form new mycelia or yeast-like forms. C. albicans infection is referred to as candidiasis, which is identified by mucosal infections of gastrointestinal epithelial cells, oropharyngeal mucosa, and vagina. Although C. albicans is the foremost infectious agent accountable for candidiasis, nonalbicans species of Candida such as Candida glabrata, Candida krusei, Candida dubliniensis, Candida parapsilosis, and Candida tropicalis have also gained weight due to their regular recovery from infected individuals.nfection . In the nfection , 3. The Despite advancements in antimicrobial drug development, it has been expected that approximately 10 million deaths will be caused by drug-resistant pathogens by 2050, which will be greater than the figure of mortalities instigated by cancer . TherefoEnterobacteriaceae, whereas date palm trees from the Middle East have so far less commonly explored for antibacterial action against other Enterobacteriaceae [Plants from this region have not been given much importance for exploration compared to their counterparts in other regions. For example, the date palm tree from the African region has been widely investigated for its antibacterial potential and mechanism against eriaceae . The extCandida infections. Based on the number of nitrogen present in the five-membered azole ring, these drugs have two main groups, namely, imidazole (two nitrogen atoms) or triazoles (three nitrogen atoms). Azoles are not fungicidal but fungistatic against filamentous fungi and yeasts that attack the ergosterol synthesis pathway in the cell wall. Ergosterol maintains the fluidity and integrity of the cell wall; hence, inhibition of ergosterol synthesis results in inhibition of fungal growth [α-demethylase encoded by the gene ERG11. As a result, the level of ergosterol needed for maintaining a normal cell membrane gets reduced [β-d-glucan synthase that results in weak cell wall and unable to resist osmotic pressure [Azoles are a class of antifungal agents that are most used against l growth , 12. Som reduced . The lan reduced . Other a reduced . Polyene reduced . 5-Fluor reduced . Caspofupressure . Allylampressure .Candida species produce biofilms on synthetic materials [Candida, accelerate drug resistance, and act out for chronic infections [Irrespective of the incessant and progressive attainments in the medical field, there are imminent risks that cannot be ignored. One such warning is the upsurge in antimicrobial resistance among pathogens due to overprescription and the promotion of resistance among cancer-curing drugs all the way through DNA mutation in cancerous cells . Moreoveaterials . Biofilmfections .Candida species is a major problem and reason for treatment failure in clinical settings, and hence, there is a need to develop newer antifungal molecules and/or modifications of existing antifungals to make them more effective and less toxic. These newer antifungals can be designed or extracted from plants, animals, or other fungi. The present review examines some of the promising alternative natural compounds that have been tested and found effective in treating Candida infections in vitro in some Middle Eastern countries. Due to inadequate reviews centering on bioactivities from plant products from the Middle East, we intend to give a discussion on plants from this region that have a variety of bioactivities and to make available evidence on the compounds that can be discovered from these plants. This is to augment our knowledge, improve modern medicine problems such as drug resistance, and achieve an alternative cure for fungal infections.The development of antifungal resistance in The Middle East is recognized as the most dried-out region on the planet and comprises Bahrain, Cyprus, Egypt, Iran, Iraq, Israel, Jordan, Kuwait, Lebanon, Oman, Palestine, Qatar, Saudi Arabia, Syria, Turkey, the United Arab Emirates, and Yemen. This part of the world has got varied physical geographies, ranging from enormous gravel and sandy deserts to highland plateaux and mountain ranges. Additionally, the climate varies depending on the season; in summer, the temperature lies typically at 38–42°C, whereas during winter, the temperature may fall to 14°C .Acantholimon, Acanthophyllum, Astragalus, Centaurea, Cousinia, Dionysia, Nepeta, Phlomis, Salvia, Saponaria, Silene, Stachys, Thymus, and Verbascum [Al-Ṭibb al-Nabawi) by Ibn Qayyim al-Jawziyya. This practice is still being used in folk medicine in the Arabian world [The Law in Medicine (Al-Qanun Fi Al-Tibb) [Candida efficacy. In Despite being the driest region in the world, it inhabits over 13,500 species of plants. The most common genera are erbascum , 23. Theerbascum . The conan world . HistoriAl-Tibb) , and untAl-Tibb) . The sigC. albicans. The plants analyzed for antibacterial activities were Mentha cervina, Ocimum basilicum, Mentha pulegium, Origanum vulgare, Salvia officinalis, Ruta graveolens, and Scirpoides holoschoenus. The GC-MS analysis of essential oils from these plant extracts revealed the presence of various bioactive compounds. Some of the major bioactive compounds were pulegone, L-linalool, 1-terpineol, 1-menthone, and eucalyptol detected in the M. cervina oil. The major components in O. basilicum essential oil were L-linalool (60.9%) and estragole (21.5%). In addition to these major compounds, other compounds were detected in lower amount such pulegone (4.2%), eucalyptol (2.2%), trans-α-bergamotene (1.5%), and so on. On the other hand, the major components detected in O. vulgare oil were 1-terpineol, sabinene, γ-terpinene, α-humulene, and α-phellandrene [M. cervina, O. basilicum, and O. vulgare were found highly inhibitory against the tested microorganisms in terms of the size of their inhibition zones. The authors of the study reported that essential oils from M. cervina, O. basilicum, M. pulegium, O. vulgare, and R. graveolens were highly inhibitory against C. albicans as the zone of inhibition was more than 30 mm; however, S. officinalis and S. holoschoenus could not inhibit the growth of C. albicans. In terms of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) as determined through the microdilution method, M. cervina essential oil was most effective against C. albicans with MIC and MFC values of 0.4 mg/ml and 0.8 mg/ml, respectively [M. cervina oil. The essential oil of M. pulegium exhibited a moderate antimicrobial activity against the strains.In a study, essential oils were extracted from seven different aromatic plants commonly found in the Asir region of southwestern Saudi Arabia and were subjected to antimicrobial and antifungal testing against various bacterial species and landrene . The essectively . The autSalvadora persica L.) is a plant associated with oral hygiene, growing primarily in Saudi Arabia and also in other regions of the Middle East [Actinobacillus actinomycetemcomitans ATCC 43717, Actinomyces naeslundii, C. albicans ATCC 90028, Lactobacillus acidophilus CCUG 5917, Porphyromonas gingivalis W50 Black, Prevotella intermedia VPI 4197, and Streptococcus mutans CCUG 11877. The ethanol extracts of twig and root inhibited the growth of C. albicans, and compounds such as N-benzylbenzamide, decane, and stigmasterol were identified to exhibit antimicrobial potential [Meswak , Fagonia indica (Shoka'a), Lawsonia inermis (Henna), Portulaca oleracea (Baq'lah), S. persica (miwak), Ziziphus spina-christi (Sidr), and Asphodelus tenuifolius (Kufer) after extraction with ethanol against C. albicans. Out of the above-mentioned plants, L. inermis and P. oleracea were found to exhibit significant anti-Candida activity with an MIC of ∼10 μg/ml. Both plant extracts were also able to inhibit C. albicans growth at its active growth phases including biofilm development and age resistance. L. inermis and P. oleracea extracts were also exhibited antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and the multidrug-resistant (MDR) Acinetobacter baumannii and Klebsiella pneumoniae [Soliman and colleagues in Sharjah (UAE) screened eumoniae .Aspergillus niger and costus extract (NCt/CE). The synthesized NCt, CE, and their nanocomposites were tested against resistant C. albicans and C. glabrata and were found to exhibit significantly stronger anticandidal activity against the examined strains. NCt/CE nanoconjugates can be used as a novel drug to control resistant pathogenic Candida strains [Candida to treat vaginal candidiasis. Four types of monofloral honey were tested such as Markh, Manuka, Qatad, and Sider, and all were inhibitory to the growth of C. albicans at 80 and 95% honey concentration. Markh and Manuka honey at 50% concentration completely inhibited the growth of C. albicans. Moreover, oil from Taif rose inhibited C. albicans completely at 2% concentration following incubation up to 48 hours. The mass spectrometry analysis revealed the presence of gallic acid and quercetin in Markh honey that are known to possess antifungal activity [Alshubaily synthesized nanoconjugates using chitosan from  strains . In a stactivity .Syzygium aromaticum) has been extracted with different organic solvents and tested for activity against C. albicans, C. glabrata, and C. tropicalis. The clove extract was reported to contain eugenol (58.8%), eugenyl acetate (23.8%), trans-Caryophyllene (14.4%), and α-Humulene (1.8%). Among all organic extracts of clove, ethyl acetate extract showed the highest inhibitory activity in terms of zone of inhibition against the test species of Candida [S. persica) root stick is widely used throughout the Arabian and Muslim world during ablution before every daily prayer as it is highly recommended for oral hygiene by Prophet Mohammad. The meswak extract (20%) was found highly effective against C. albicans and Enterococcus faecalis when treated for 6 hours or more, although the same extract was ineffective when treated for 1 hour [A common spice, clove , 1H NMR (nuclear magnetic resonance), 13C NMR, and mass spectral analysis. The isolation leads to the novel compound called isopimpinellin with the molecular formula C13H10O5. The compound was found to possess encouraging antimicrobial activity in terms of MIC against Bacillus subtilis (100 μg/ml) and Klebsiella pneumoniae (75 μg/ml), Aspergillus niger (150 μg/ml), and Candida albicans (75 μg/ml). Furthermore, the novel compound (isopimpinellin) was found to inhibit fungal biomass. Moreover, isopimpinellin exhibited antibiofilm activity against the uropathogenic strains [The genus orldwide . Many sptivities , 65, 66.ive days . Haplophinations . The act strains .Deverra tortuosa is widespread in Al Widyan region, particularly in the valley of Arar, Saudi Arabia [D. tortuosa plant is eaten by various herbivorous animals such as camels. It has been used in traditional medicine for the treatment of constipation, hypertension, and bites [D. tortuosa essential oil is Apiol (65.7 to 74.4%) and has good antioxidant activity. Its essential oil exhibited strong activity against yeasts Malassezia spp and Candida krusei with MIC values ranging between 2.8 mg/ml and 27 mg/ml [Psiadia punctulata collected from Saudi Arabia was investigated for its antimicrobial activity against S. aureus and C. albicans. The extract was reported to possess antimicrobial activity against S. aureus with an MIC of 180 μg/ml and activity against C. albicans with an MIC value of 130 μg/ml. Furthermore, the isolated 3′,4′,5,7-tetramethoxyflavone was found to inhibit biofilm formation of S. aureus and C. albicans by 50 and 90%, respectively, at 40 μg/ml concentration [The perennial shrub i Arabia . The D. nd bites . The maj27 mg/ml . The surntration .S. aureus (ATCC 6538), P. aeruginosa (ATCC 15442), E. coli (ATCC 11229), and C. albicans ATCC 10231 were determined by disc diffusion and broth dilution methods and found to exert a moderate antimicrobial effect on the tested microorganisms with MIC values ranges between 128 and 512 μg/ml [The Turkish flora has been estimated to contain 11,000 taxa out of which 1,280 have been in use as traditional medicines , 69. A s12 μg/ml .Mentha longifolia, Mentha piperita, Prangos ferulacea, Galium verum, Salvia limbata, Artemisia austriaca, Plantago lanceolate, and Urtica dioica were extracted with methanol and chloroform and subjected to evaluation for their anti-Candida activities. In total, 102 Candida species were tested, of which, 99 were human-pathogenic isolates . Three standard strains were also used in the study as reference. Plants extracted with chloroform did not show any inhibitory effect against any of the Candida isolates; however, methanol extracts of these plants were reported to possess strong anti-Candida activity [Some common folk medicinal plants of Turkey such as activity .Centaurea have been widely used in traditional medicine in Turkey for various diseases such as dandruff, diarrhea, inflammation, digestion, fever, and infections [Centaurea baseri essential oil and crude extract showed the presence of hexadecanoic acid (42.3%), nonacosane (8.2%), and heptacosane (8.0%) that were the main components of its essential oil. The extract and essential oil of C. baseri were found to exhibit a strong inhibitory effect against Candida utilis and Bacillus cereus with MIC values of 60 and 47 μg/ml, respectively. The extract of C. baseri was also reported to have highly selective cytotoxic properties against various cell lines such as MCF-7, PANC-1, A549, and C6 glioma cells [Many species of the genus fections . Chemicama cells .Anthemis stiparum that is commonly used as a medicinal herb for various ailments was analyzed for its chemical composition, phenolic and flavonoid contents, and antimicrobial and antibiofilm activities. Chemical analysis of its essential oil revealed the presence of germacrene D (11.13%), t-cadinol (11.01%), camphor (6.73%), spathulenol (6.50%), and isoamyl salicylate (6.45%). The methanolic extract constituted 13.6% and 5.9% of pyrocatechol and quercetin, respectively. The methanolic extract was reported with better antimicrobial activity than the essential oil of A. stiparum against S. aureus (ATCC® 25923) and Bacillus subtilis (ATCC® 6633), with an MIC of 1.56 mg/ml. The methanolic extract was found to inhibit Candida albicans (ATCC® 10239) adhesion by 80% at 6.25 mg/ml concentration [In a study, ntration .Arum maculatum, Datura stramonium, Geranium asphodeloides, and Equisetum telmateia were highly inhibitory against S. epidermidis, E. coli, and C. albicans [In another study, several plant species used as traditional medicine in Sakarya province (northwest Turkey) were studied for their antimicrobial activities following extraction with petroleum ether and ethanol. The authors of the study reported that petroleum ether extracts of plants such as albicans .Papaver rhoeas is a red poppy, which is known as “gelincik” in Turkey. The species is found all over Turkey and used for various medicinal purposes such as cough syrup for children and tea for insomnia, sedative, and pain relief [P. rhoeas samples from different parts of Turkey and examined for their alkaloid content as well as their antimicrobial activities against S. aureus and C. albicans. The samples were reported to contain 12 different types of alkaloids belonging to proaporphine, aporphine, promorphinan, protopine, and rhoeadine groups. The most significant antimicrobial activity was observed with the alkaloid aporphine (roemerine) against S. aureus and C. albicans with MIC values as low as 1.22 μg/ml and 2.4 μg/ml, respectively [n relief . Coban aectively .Ebers Papyrus, written in 1550 BC with 876 prescriptions made of 328 different ingredients derived from many plant species. Notable medicinal plant species still in use since ancient civilizations of Egypt are Artemisia absinthium, Acacia nilotica, Balanites aegyptiaca, Bryonia sp, Hyoscyamus muticus, Myrtus communis, Onopordon sp, aloe, gums, myrrh, pomegranate, colocynth, linseed, cumin, and Ziziphus sp [A total of 2,174 plant species have been recorded in Egypt; 121 of them are known for their use for medicinal purposes . Historiiphus sp .Clerodendrum belongs to the family Lamiaceae and is found in many countries including Egypt. This plant has been reported to be used in treating various diseases and ailments [Clerodendrum chinense from Egypt were reported to contain phenylpropanoid glycosides , flavonoid (hispidulin), cyclohexylethanoids (cornoside and rengyolone), and icariside B5 [Clerodendrum have been shown to exhibit strong antimicrobial activity [C. chinense and C. splendens were highly inhibitory against Plasmodium falciparum and Trypanosoma cruzi; however, the same extracts were found moderately effective against C. albicans [The plant ailments , 75. Theiside B5 . Severalactivity . Chlorofalbicans .Calocedrus decurrens, Cupressus sempervirens, and Tetraclinis articulata. The extracted essential oils were subjected to antimicrobial activity testing against Gram-positive, Gram-negative bacteria, and Candida species. δ-3-Carene (43.1%), cedrol (74.0%), and camphor (21.2%) were, respectively, the major components of essential oils of C. decurrens, C. sempervirens, and T. articulata. These essential oils exhibited strong anti-Candida activity against several species of Candida such as C. albicans, C. glabrata, C. krusei, and C. parapsilosis [Ibrahim and coworkers have extracted essential oils from aerial parts of the plants psilosis .C. albicans, S. aureus, and E. coli. The antimicrobial activities of all four formulations  against C. albicans were comparable to the commercially available gentamycin ointment. On the other hand, the formulations performed better against bacterial species compared to the gentamycin ointment [Ceratophyllum demersum, Eichhornia crassipes, Potamogeton crispus, and Potamogeton pectinatus were tested against C. albicans and C. tropicalis. The aqueous extract of P. crispus was reported to be the most inhibitory against both Candida species, followed by the aqueous extract of E. crassipes. Although chloroform extracts were also effective against Candida species, the ethanol and methanol extracts were only moderately effective [An antimicrobial study was conducted using formulations prepared with extracts (15%) of henna, pomegranate, and myrrh as well as their blends, and the activity was compared with the marketed gentamycin ointment. The extract formulations were found to exhibit strong antimicrobial activity against ointment . The aquffective .Chlorophyllum molybdites, Ganoderma xylonoides, Trametes cingulate, Agaricus bernardii, Agrocybe pediades, Coriolopsis polyzona, Pycnoporus sanguineus, and Trametes lactinea were reported with strong inhibitory effect against bacterial species tested. On the other hand, methanol extracts of C. molybdites, G. xylonoides, and P. sanguineus were found with considerable antifungal activity [Ganoderma pfeifferi (a basidiomycete) was examined for chemical composition and antimicrobial activity. The antimicrobial effects of volatile oil were tested against 5 bacteria and 2 Candida species  using disc diffusion and broth microdilution methods. Ninety percent of volatile oil corresponded to 4 compounds, namely, amyl-vinyl-carbinol (73.6%) followed by 1-octen-3-ol acetate (12.4%), phenylacetaldehyde (3.0%), and 6-camphenol (1.5%). The volatile oil was more inhibitory to Gram-positive bacteria than to Gram-negative ones. The oil exhibited strong antimicrobial activity against C. albicans with an MIC of 600 µg/ml [In a study from Yemen, the antimicrobial activities of different basidiomycete species were investigated following extraction with dichloromethane, methanol, and aqueous extracts. The methanol extracts of activity . The vol00 µg/ml .Stachys yemenensis was analyzed for its chemical composition and antimicrobial activity. The essential oil consisted was α-phellandrene (13.9%), β-phellandrene (11.7%), elemol (12.0%), spathulenol (6.7%), β-eudesmol (5.0%), α-eudesmol (4.75%), and squalene (4.8%). The essential oil in concentration ranged between 0.001% (v/v) and 2.5% (v/v) was tested by using the broth dilution method against E. coli ATCC 35218, S. aureus ATCC 43300, and two clinical strains, C. albicans and C. glabrata. MIC value against E. coli strain was 0.15% (v/v) in hydro-distilled extract and 0.3% (v/v) for supercritical fluid extract. For S. aureus, the MIC values were 0.6% (v/v) in supercritical fluid extract and 2.5% (v/v) in hydro-distilled. The hydro-distilled and supercritical fluid extract of S. yemenensis essential was not strongly inhibitory against the C. albicans and C. glabrata as MIC values were higher than 2.5% (v/v) except for C. glabrata, for which the MIC of the supercritical fluid extract was 2.5% (v/v) [The essential oil from the leaves of 5% (v/v) .μg/ml. The butanol extract of Rosa damascena inhibited 100% of Salmonella typhimurium and Bacillus cereus at concentrations of 62.5 and 250 μg/ml, respectively. Furthermore, the butanol extract of Narcissus tazetta and aqueous extract of Rosa damascena receptacles were highly inhibitory against C. albicans at a concentration of 125 μg/ml [Jordan although a small country contains around 2,500 plant species in which 285 species have been described as medicinal plants . Talib a25 μg/ml .Anthemis, which comprises nearly 210 species. It is distributed across Europe, Southwestern Asia, Northern and Northeastern Africa, Southern Arabia, and tropical East Africa [Anthemis have been in use in traditional medicine since the time of the Roman Empire [Anthemis have tremendous antimicrobial potential that could be correlated with their phenolic and flavanoid compositions [Anthemis palestina is mostly found in the middle and northern mountainous regions of Jordan and is known as Palestine chamomile due to its resemblance to Roman and German chamomiles [A. palestina. They extracted oil from A. palestina through hydro-distillation and tested against many bacterial and Candida species including C. albicans, C. glabrata, and C. krusei. The extracted oil was reported to possess a broad spectrum of antimicrobial activity, especially against Gram-positive bacteria. Furthermore, the oil was also inhibitory for C. albicans, C. glabrata, and C. krusei having MIC values ranging between 48 and 95 μg/ml [One of the most important genera of the family Asteraceae is t Africa . Variousn Empire , 80. Recositions . Anthemiamomiles . Bardawe95 μg/ml .Artemisia judaica is another plant widely used in Jordanian folk medicine. A. judaica has been reported for the treatment of inflammation and infections caused by fungi, bacteria, and viruses [A. judaica when extracted with a mixture of water and ether was found to inhibit Klebsiella aerogenes, P. aeruginosa, and S. aureus [A. judaica oil were piperitone (30.4%), camphor (16.1%), and ethylcinnamate (11.0%). Cryptococcus neoformans was found to be highly susceptible to the oil from A. judaica with an MIC value of 0.16 μl/ml. The interesting part of the oil from A. judaica was its strong inhibitory effect on germ tube formation in C. albicans. Eighty percent inhibition of filamentation was recorded at a concentration of 0.16 μl/ml. Moreover, the oil successfully disrupted the preformed biofilms in terms of reducing the amount of biomass attached [ viruses . A. juda. aureus . It has . aureus . Abu-Darattached .Candida species. At least 20 different species are known to inhabit the oral cavities of normal and immunocompromised persons. Adwan and coworkers carried out a study to investigate and compare anti-Candida activities of nine different commercial toothpaste with and without herbal extracts against 45 oral and nonoral Candida isolates [Candida isolates, but the toothpaste containing herbal extracts were found to exhibit higher anticandidal activity [Scolymus maculatus (golden thistle) is believed to be curative for many diseases in Palestinian region. Different extracts of S. maculatus were investigated for their chemical composition and antimicrobial activity. The extracts were reported to contain stigmasterol, γ-sitosterol, lupeol, lupeol acetate, and β-amyrin among many others. MICs of S. maculatus extracts were determined against S. aureus, S. typhimurium, and C. albicans. Different organic extracts of S. maculatus exhibited an MIC of 500 μg/ml against bacterial isolates and C. albicans, whereas aqueous extract was having a higher MIC value [The human oral cavity is a habitat for a large number of microorganisms including various isolates . Althougactivity . The plaIC value .Nepenthes khasiana (a carnivorous plant). The antifungal activities of droserone and 5-O-methyldroserone were compared. The antifungal activity revealed that droserone was more active when used as an element of the pitcher liquid or as a purified compound. When tested against Candida and Aspergillus spp., the inhibitory and fungicidal effects were observed at a significantly lower concentration than the cytotoxic amount in the cells of a human embryonic kidney cell line, 293T. These naturally occurring naphthoquinones may lead us to develop newer antifungal drugs with reduced toxicity [Many plants, particularly carnivorous plants, produce naphthoquinones that have been reported for antifungal activities . In a sttoxicity .Lavandula (lavender) have been in use for a long time for medicinal purposes in Palestine region. Three species are native to the Palestine region, namely, Lavandula pubescens, Lavandula stoechas, and Lavandula coronopifolia [L. pubescens has been reported to possess strong antibacterial activity in vitro against many types of bacteria such as Salmonella enterica, S. aureus, Micrococcus luteus, E. faecalis, and E. coli [L. pubescens) have been utilized for centuries as herbal medicine in the region [L. pubescens revealed the presence of many compounds in which carvacrol (a type of monoterpene) was reported as the most abundant (65.3%). The essential oil from L. pubescens has been shown to demonstrate strong antibacterial activity against S. aureus with 95.7% inhibition. The essential oil was also found to exhibit strong inhibitory activity against C. albicans with an MIC value of 0.47 μl/ml. Furthermore, the oil was also reported to possess antidermatophyte activity against Microsporum canis, Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton floccosum [Various species of the genus opifolia . Severalopifolia , 88, antopifolia . They aropifolia . The ess E. coli , 91. Mane region . The phyloccosum .Scrophularia sp) is a commonly found plant in Iran, spread across the mountain ranges and deserts [S. striata) against oral pathogens. A systematic review of the medicinal properties of Dracocephalum kotschyi and its significance in Iran has highlighted potential health benefits of this plant from this region. The plant has been well-documented for its antioxidant, antitumor, and antimicrobial efficacies against C. albicans [C. albicans isolates from patients and reported the anti-Candida activity of Syzygium aromaticum extract as significantly better than the activity of nystatin (P < 0.001). Achillea millefolium (common name: yarrow), which belongs to the family Asteraceae, is traditionally used for various female genital disorders such as inflammations, infections, and dysmenorrhea [C. albicans as well as resistance against other fungal pathogens [A. millefolium (2%) extract in the cream formulation in comparison with clotrimazole vaginal cream in patients with vulvovaginal candidiasis. The study was conducted on 80 women diagnosed with vulvovaginal candidiasis. Half of the women were given clotrimazole 1% vaginal cream, and the remaining women were given vaginal cream containing the aqueous extract of A. millefolium for 7 days. The results were suggestive that vaginal cream containing A. millefolium could reduce the complaints of vulvovaginal candidiasis [D. kotschyi was analyzed using GC-MS. In cultivated plants, major compounds of essential oil were α-pinene (13.66%), (E)-citral (12.89%), neral (11.25%), methyl geranate (8.66%), limonene (8.33%), campholenal (6.22%), and geraniol (5.69%). On the other hand, a stark difference was noted in the composition of EO in naturally grown plants in which two main compounds, cyclohexylallene (52.63%) and limonene (35.88%), were predominant. The antimicrobial activities of this plant were determined against 12 different microorganisms. The MIC value against Bacillus subtilis was 31.25 μg/ml; the MIC was lower than that obtained by treating the same strain with Rifampin. The EO obtained from wildly growing D. kotschyi was highly inhibitory against C. albicans  lower than that obtained treating the yeast with Nystatin [Peganum harmala (wild rue) is used as a topical antifungal agent for treating many infections in Iran. In a study, P. harmala was investigated for its inhibition of C. albicans biofilm formation. C. albicans (27) were collected from women with vaginitis and grown to form biofilm and then P. harmala EO was applied as an antibiofilm agent. The researchers noted that P. harmala EO in the concentration of 12 μg/ml strongly inhibited biofilm formation. Moreover, at lower concentrations (10 and 6 μg/ml), the essential oil of P. harmala was also effective in controlling biofilm formation albeit weakly [The figwort  is a plant native to the Iranian region and has been documented to possess antifungal assets [C. glabrata and C. krusei by Eslami et al. [C. krusei is more sensitive to the chicory ethanol extract compared to C. glabrata. They found that the antifungal activity of chicory leaf extract is due to the presence of compounds such as lactucin, lactucopicrin, deoxylactucin, and α-1,3-dihydrolactucin. The compounds identified were sesquiterpenes that inhibited the growth of yeast by disrupting the membranous structure of the Candida cells [Chicory , δ-3-carene (22.1%), limonene (4.6%), and α-terpinolene (4.5%) were the main components comprising 79.8% of the oil. The methanol extract of C. sempervirens was highly inhibitory to the growth of the bacteria studied. The essential oil and methanol extract of C. sempervirens eradicated the biofilm from the surface and thus can be employed as a natural preservative in food and pharmaceuticals [ceptives . Its leap-making . In a steuticals .in vitro antimicrobial potential of several water and methanol extracts obtained from diverse parts of 27 ethnic wild plants used in Lebanese folk medicine. The microorganisms under study were Escherichia coli, Proteus sp, Pseudomonas aeruginosa, Shigella dysenteriae, Salmonella enteritidis, Salmonella typhi, Staphylococcus aureus, Streptococcus faecalis, and Candida albicans. Out of 39, only 1 water extract from Alchemilla diademata whole plant exhibited antimicrobial activity against Staphylococcus aureus. However, methanol extracts of 10 other plants were successful by showing susceptibility to test organisms such as Achillea damascena whole plant (88.8%), Anthemis scariosa flowers (88.8%), Cirsium sp whole plant (88.8%), Centaurea ainetensis flowers (88.8%), Hieracium sp whole plant (88.8%), Origanum libanoticum whole plant (99.9%), Ranunculus myosuroides whole plant (88.8%), Nepeta curviflora leaf (88.8%), N. curviflora stem, and Verbascum leptostychum flower (99.9%). They concluded that the antimicrobial activities of plants were more evident in methanol than water extracts of the indigenous plants of Lebanon.In a study, Barbour et al.  evaluateAvicennia marina, Lotus halophilus, Pulicaria gnaphaloides, Capparis spinosa, Medicago laciniata, and Limonium axillare exhibited varied levels of antimicrobial activities against E. coli, P. aeruginosa, B. cereus, S. aureus, C. albicans, and A. flavus [Cressa cretica L. was highly effective against Penicillium citrinum and Candida albicans. Their study also highlighted that diffusible metabolites of Heliotropium curassavicum have marked inhibitory effects against these pathogens. However, chloroform extract of Emex spinosa exhibited strong activity against Alternaria alternata and Saccharomyces cerevisiae. However, the highest growth inhibition was recorded for Fagonia indica against Penicillium citrinum.In a study from Qatar, aqueous, ethanol, and butanol crude extracts of the aerial parts of ten native plants viz. . flavus . On the . flavus  against Tribulus terrestris L., an Iraqi medicinal plant, were studied by Al Bayati and Al Mola [Candida albicans. The extracts from the different parts of the plant were potential antimicrobials against the majority of microorganisms. The ethanol extract from the fruits was the most active against Gram-positive and Gram-negative bacteria and showed the highest antifungal action against C. albicans . Three types of dates from Oman  have also been described to possess strong antimicrobial activities [Organic and aqueous extracts from fruits, leaves, and roots of  Al Mola  for theitivities . The anttivities .Candida strains. Many researchers have reported promising anti-Candida activities of aqueous and organic extracts prepared from different parts of several medicinal plants. In this review, it has been demonstrated that plants from the Middle Eastern region are competent to show potential antimicrobial, especially anti-Candida activities. Studies have also reported major bioactive compounds that could be developed as a drug candidate either alone or potentiating the efficacy of existing antifungals by reducing the side effects. We have compiled here the Middle Eastern plant extracts/oils/products as a potential remedy for the challenges encountered in the clinical setting, such as the surge in drug resistance. We hope this work could facilitate more research to explore plants from this region to reveal the precise mechanisms of action. A larger number of studies have been conducted in vitro, and only fewer studies have been carried out in humans, so there is a great need and scope for further improvement in studies via clinical trials in treating Candida infections.In the Middle East region, using plant materials as medicine has a very long history. Many of the current local medicinal plants were found described on clay tablets from the Mesopotamian civilizations such Sumerians, Assyrians, Akkadians, and Hittites . The pub