Honeybees are one of the most marvelous and economically beneficial insects. As pollinators, they play a vital role in every aspect of the ecosystem. Beehive products have been used for thousands of years in many cultures for the treatment of various diseases. Their healing properties have been documented in many religious texts like the Noble Quran and the Holy Bible. Honey, bee venom, propolis, pollen and royal jelly all demonstrated a richness in their bioactive compounds which make them effective against a variety of bacterial strains. Furthermore, many studies showed that honey and bee venom work as powerful antibacterial agents against a wide range of bacteria including life-threatening bacteria. Several reports documented the biological activities of honeybee products but none of them emphasized on the antibacterial activity of all beehive products. Therefore, this review aims to highlight the antibacterial activity of honey, bee venom, propolis, pollen and royal jelly, that are produced by honeybees. Apis, which is the Latin word for ‘’bee’’. These marvelous social insects live in a well-organized community and belong to the order Hymenoptera and to the family Apidae [Apis mellifera [Honeybees, also known as the ‘’Golden insect‘’, belong to the genus y Apidae . Honeybeellifera . Since aellifera . Egyptiaellifera . Apitherellifera . As for ellifera ,7,8,9,10ellifera ,11,12. Mellifera ,14,15. Aellifera , anti-agellifera , antitumellifera ,19 anti-ellifera , and neuellifera . Hence, Apis mellifera) [Honey, one of the most well-known and highly valued natural products, is produced by honeybees (llifera) . Since allifera) . Apis mellifera is characterized by a low pH level ranging between 3.53 and 4.03 [Honey is a naturally occurring supersaturated sugar solution made up of a complex mixture of carbohydrates (77–86%), with fructose and glucose being the most abundant. It has a low amount of water (around 20%) which gives honey several of its attributes such as high viscosity and high osmotic pressure. It also contains numerous minor but essential components including proteins, enzymes , organic acids , amino acids, lipids, vitamins, minerals and many others. Furthermore, honey from and 4.03 ,24,25,26and 4.03 ,28,29.2O2) pathway, H2O2 is formed by glucose oxidase, a carbohydrate metabolizing enzyme added to nectar by bees. As indicated by prior studies, the enzymatic oxidation of glucose is responsible for the antimicrobial activity of honey [2O2 was removed by catalase [The antibacterial effect of honey is mediated by two mechanisms: a peroxide-dependent and a peroxide-independent pathway. In the hydrogen peroxide  [L. scoparium [Stephalococcus aureus, Bacillus subtilis, and Paenibacillus larvae, the main cause of the ravaging American bee larvae disease [As for the peroxide independent pathway, the physicochemical properties consisting of high viscosity and high sugar content are the two factors that confer honey its antibacterial effect. In fact, the extraction of moisture from the surrounding environment causes bacterial dehydration by osmotic pressure. In a previous study of infants with gastroenteritis where glucose given in oral rehydration solution was substituted by honey, the recovery time of patients was significantly reduced . This isflowers) . Manuka coparium ,40. Thiscoparium ,42,43. I disease . Escherichia coli strains with ampicillin-resistance gene (β–lactamase) were found to be sensitive to honey [E. coli growth and had a significant amount of non-peroxide antimicrobial activity [2O2 dose-dependent [With the emergence and spread of resistant pathogens, the properties of honey could be potentially clinically relevant. Honey demonstrates a wide range of antibacterial activity encompassing both Gram-negative and Gram-positive bacteria. In a previous study, to honey . In addiactivity . Anotheractivity . These fependent . Helicobacter pylori, causative agent of peptic ulcers [In addition honey has been discovered to be a powerful inhibitor of c ulcers . Staphylococcus pseudintermedius which cause serious infections in domesticated animals and can be transmitted to their owner. They demonstrated that Manuka honey is not just effective against novel multidrug-resistant S. pseudintermedius isolates, but additionally works in tandem with clinically important antibiotics and reduces its virulence [As Manuka honey possesses an exceptional antibacterial activity, Brown et al. tried it against irulence . Salmonella interitidis to the intestinal epithelial cells in vitro and has demonstrated that honey at dilutions 1:8 diminished the bacterial adherence from 25.6 ± 6.5 (control) to 6.7 ± 3.3 bacteria/epithelial cell [A previous study has assessed the capacity of honey to prevent the adherence of ial cell .Streptococcus mutans from forming a biofilm, implying that honey could help to minimize oral pathogens in dental plaque [S. aureus (MSSA), MRSA, and Pseudomonas aeruginosa, with bactericidal rates of 63–82%, 73–63%, and 91–91%, respectively, which were higher than the effect of widely used single antibiotics [Furthermore, another study found that a 10% concentration of honey could prevent oral bacteria like l plaque . Honey wibiotics .Burkholderia cepacia, which is responsible of pulmonary infections especially in patients with cystic fibrosis and chronic granulomatous disease. Twenty strains of B. cepacia isolated from the sputum of patients with cystic fibrosis were screened for their susceptibility to eight antibiotics and two types of honey (with and without peroxide activity). All strains were resistant to the antibiotics tested but susceptible to honey at concentrations less than 6% (v/v) [P. aeruginosa and against E. coli at different concentrations . All honey samples displayed an inhibitory effect on the growth of P. aeruginosa and E. coli for concentrations above 2.5%. Only four samples were still effective at a concentration of 2.5%: one against E. coli and three against P. aeruginosa. No activity was observed at concentrations below 2.5% [In another study, honey demonstrated antibacterial activity against 6% (v/v) . In addilow 2.5% . Streptococcus pyogenes and S. aureus. Four honey samples were taken from beekeepers and tested at various concentrations , then compared to standard antibiotics. Honey samples exhibited antibacterial activity with a diameter of the zone of inhibition (ZDI) ranging between 0 and 46 mm for S. aureus and between 0 and 44 mm for S. pyogenes [E.coli, Klebsiella pneumonia, Salmonella spp., etc… [The antimicrobial effect of honey has been tested against two Gram-positive organisms: pyogenes . Other sp., etc… ,56.Porphyromonas gingivalis, a Gram-negative bacterium. A previous study has shown that honey helps to prevent periodontal disease by killing anaerobic bacteria [Honey can be utilized to treat a wide range of oral diseases, including periodontal disease which is caused by bacteria . It is worth mentioning that the antibacterial activity of honey differs widely depending on the plant source ,54. AlsoE. coli and S. aureus were exposed to different honey samples, physiological changes in membrane integrity and polarization occurred. Honey also caused a major metabolic disturbance in S. aureus as a primary physiological consequence [Fuertes et al. have explored the repressive mechanism of honey on the growth of bacteria. They found that when sequence . E. coli and P. aeruginosa. None diluted honey and its 1:2 to 1:6 aqueous dilutions had 100% and 96.4% activity against P. aeruginosa and E. coli, respectively. Gentamicin, on the other hand, showed lower antibacterial activity when used at concentrations of 8.0 and 4.0 g/mL [Comparing the antibacterial potency of honey to that of antibiotics was also reported. A recent study compared the activity of three honeys with gentamicin against 4.0 g/mL . Combining honey with other antibacterial compounds increases their inhibitory rate against microorganisms. In fact, when honey was combined with the gentamicin, the killing rate increased to more than 92–93% while the killing rate was around 77% for gentamicin alone and around 45% for honey alone . AnotherH. pylori infection in a group of 150 dyspeptic patients [Concerning clinical trials on honey, Blaser et al. were reported a full recovery in seven consecutive patients with MRSA infected or colonized wounds. Alternatively, antiseptics and antibiotics had previously failed to eradicate infection-related symptoms . Furtherpatients . Also, ipatients .Studies used different methods to measure the antibacterial activity of honey. The most common methods were agar diffusion assay and serial dilution approach in microtiter plates. The first method, while being effortless and quick in performance, presents many limitations: (1) difficulty in loading a specific volume of the product sample into the agar wells due to the high viscosity of honey, (2) diffusion issues with active components such as defensin-1 and glucose oxidase, which have a large molecular weight across the agar matrix, (3) low reproducibility, (4) difficulty in comparing the results with those of other authors, (5) inability to discriminate between bacteriostatic and bactericidal activity. For the second method, serial dilution assay, the bacteriostatic (MIC) and bactericidal activity (MBC) of examined honey samples can be determined quantitatively using this approach in contrary to that of the first method. The only challenge that this method presents is the preparation of honey output solution .In light of these findings, honey appears to exert wide antibacterial activity against a variety of microorganisms. It has been suggested in the literature that these properties make the development of honey-resistance unlikely and extrapolate that honey may have an important clinical utility in the treatment of antibiotic-resistant bacteria ,71,72. AApis mellifera venom is an odorless and transparent liquid that is made up of 88% of water and only 0.1 µg of dry venom [2 (PLA2) and hyaluronidas [2, being the most toxic bee venom peptide [Apis mellifera purebred as well as hybrid was tested against five bacterial strains. The results showed that BV displayed an antibacterial activity against all five bacterial strains. Other studies proved that BV can be used as a complementary antimicrobial agent against pathogenic bacteria even if it is collected by different methods  [Borrelia burgdorferi is known to be the main cause of Lyme disease. Researchers found that both BV and melittin possess significant effects on all the morphological forms of B. burgdorferi, even on antibiotic resistant attached biofilms. However, antibiotics when administrated alone or combined together had limited effects [Salmonella. Sixteen Salmonella strains isolated from poultry were tested against apitoxin (Bee venom). In 14 of the 16 tested strains, apitoxin reduced the biofilm formation and destroyed the preformed biofilm by 47%. Also, apitoxin increased the mobility of the bacteria and the MIC varied between 1024–256 μg/mL [Salmonella gallinarum. The results showed that BV increased the antibody production against formalin-killed S. gallinarum [Bee venom (BV) is a widely recognized toxin secreted by female worker bee’s poison glands as a protection mechanism . Althougry venom . The dryuronidas ,82. Amonuronidas . Another peptide . These t peptide . This mo peptide . Several peptide ,88. Indechamber) . Additio effects . Food-bollinarum .S. aureus and E. coli. The results proved BV to be active in killing both bacterial strains. At 10 × MIC, the regrowth of E. coli was not observed at 18 h while For S. aureus at 5 × MIC [B. subtilis. Alternatively, Gram-negative strains were found to be more resistant to BV (MIC 60 to >500 µg/mL). This can be explained by the nature of the bacterium cell membrane [2, which is also presented in abundance in the crude BV can disrupt the cell membrane of Gram-negative bacteria indirectly by hydrolyzing phospholipids enzymatically. Other works reported the antibacterial activity of the crude BV and its two main biopeptides melittin and PLA2 against oral pathogen responsible of tooth decay: Lactobacillus casei, Streptococcus salivarius, S. mutans, Staphylococcus mitis, Staphyslococcus sobrinus, Staphylococcus sanguinis and Enterococcus faecali. Melittin was twice as active as the crude BV against the tested bacteria (4 to 40 µg/mL). As for PLA2, it was only active against L. casei at >400 µg/mL [2 did not show any effect on the same strains [E. coli but the latter was more efficient [E. coli, P. aeruginosa and MRSA. The two derived peptides showed strong antibacterial activity against reference strains of the three multidrug resistant strains compared to the indigenous melittin via membrane alteration and damage [Others previous studies focused on the antibacterial effect of bee venom against additional type of bacteria such as  5 × MIC ,93. Regamembrane ,93; the membrane . PLA2, w00 µg/mL ,96. Simi strains . Also, i strains . Likewisfficient . Intrigud damage .Acinetobacter baumannii bacterial strains. Results showed an inhibition of the bacterial growth of MDR-A. baumannii strains with MIC value 31.25 mg/mL [A. baumannii strains. The combination of melittin–ceftazidime and melittin–doripenem was administrated to MDR-P. aeruginosa strains. This caused a decrease in melittin dosage which led to a decrease in melittin cytotoxicity. Therefore, the combination of melittin–doripenem (for A. baumannii) and melittin–doripenem with melittin– ceftazidime (for P. aeruginosa) at their MIC dose might serve as a promising therapeutical treatment against MDR bacteria [On the other hand, antimicrobial treatments are becoming less efficient by the day due to the increasing number of multi-drug resistant (MDR) bacteria as well as bacterial biofilms which are very hard to treat. Between 317 positive specimens for bacterial growth, 124 were MDR isolate of Gram-negative and Gram-positive bacteria. BV demonstrated a complete growth inhibition percentage (100%) against all tested MDR-isolates with a wide range of MICs and MLCs concentration-ranging between 3.125–50 μg/mL and 6.25–100 μg/mL, respectively against all MDR-GNB and GPB one . The ant25 mg/mL . Melittibacteria .β-lactam antibiotics against A. baumannii was evaluated by means of the broth microdilution method. The MIC value of melittin was 4 µg/mL and results showed inhibition of bacterial growth. Furthermore, FIC indices for combination of melittin with co-amoxiclav, ceftazidime and imipenem showed a synergetic effect [The investigation of BV in pre-clinical and clinical application is still very slow despite its efficacity when combined with other drugs to treat different types of bacteria. Researchers found that BV and melittin combined with other antibiotics like vancomycin, oxacillin, and amikacin yielded fractional inhibitory concentration (FIC) indices fluctuating between 0.24 and 0.5. Apitoxin and melittin tested against 51 strains of bacteria exhibited strong antibacterial activity against both Gram-negative bacteria  and Gram-positive . Moreover, a strong anti-MRSA and anti-VRE activity was shown by BV and melittin at MIC values of 6–800 μg/mL . When coc effect .2 exhibit a very promising antibacterial activity against several pathogenic bacterial strains even against multidrug resistant bacteria. However, one should always keep in mind that these biopeptides are venomous and might act as toxic agents if administrated the wrong way. Melittin is the most toxic component in BV. While being responsible for the majority of the pain associated with bee stings, it only induces a minor allergic reaction. Contrary to PLA2 which is the most allergenic and immunogenic protein in bee venom [All of the above- mentioned information confirm that BV and its two major compounds melittin and PLAee venom ,107. Beeee venom . The ideee venom ,110.Many clinical studies on bee venom have shown promising results. Acne, one of the most common problems among teenagers, is usually treated with antibiotics to kill the causative bacteria but in some cases bacterial resistance can occur . In a clAmong the many products of honeybees, propolis is considered as one of the most interesting products utilized as the main defensive element and building block in the hives. Propolis is used by bees to fill the cavity in the walls of the hive, it is also used to repair combs and strengthen its thin borders. On the other hand, propolis can mummify intruders that cannot be transported outside thus preventing their decay . Since ap-coumaric acid) is considered as one of the most important phenolic compounds found in propolis. Propolis ethanolic extract showed higher antibacterial activity against MRSA in comparison with hexane extract due to the higher concentration of Artepillin C in ethanolic extract [P. gingivalis, a bacterium responsible for periodontal diseases. Artepillin C showed a bacteriostatic effect with membrane blebbing [p-coumaric acid, artepillin-C, drupanin and kaempferide alongside an antibacterial activity against Listeria monocytogenes, Enterococcus faecalis, S. aureus and Staphylococcus saprophyticus [S. aureus [S. mutans, S. sobrinus, S. aureus, E. faecalis, L. monocytogenes, P. aeruginosa and K. pneumoniae [Artepillin C  and pterocarpans. Nepalese propolis exhibited its highest antibacterial activity against . aureus . The ethPP alone . The geod Europe . EEP samd Europe .Lactobacillus acidophilus, Prevotella oralis, P. gingivalis, Fusobacterium nucleatum, Peptostreptococcus anaerobius, Actinomyces naeslundii and Veillonella parvula [Porphyromona, Fusobacterium, Propionibacterium, Clostridium, Prevotella, Actinomyces and Bacteroides species [The number of studies concerning the antibacterial effect of propolis against anaerobic bacteria remains limited. However, it has been presented that propolis is more active on Gram-positive anaerobic bacteria than on Gram-negative ones. Propolis showed antibacterial activity against  parvula . Moreove species ,132.S. pyogenes, VRE ATCC 51299 and MRSA NCTC 10442 [Propolis displays different antibacterial mechanisms such as inhibition of cell division, collapse of membranes and cell walls of the microbial cytoplasm, decreasing bacterial motility, enzymatic inactivation, bacteriolysis and inhibition of protein synthesis . All theTC 10442 . Also, sTC 10442 . EEP hasTC 10442 .Propionibacterium acnes and Staphylococcus epidermidis, as well as being effective against facial acne [P. gingivalis [Propolis is widely used in medicine due to its various bioactivities. The effect of topical propolis extract was tested on facial vulgaris acne where subjects were treated with a topical solution of ethanolic extract of propolis. This solution showed a significant bacteriological activity on ial acne . To testial acne . In anotngivalis .Pollen of various flowers is collected by field bees and agglutinated by bees enzymes secreted by the salivary glands and by the nectar. Then, the pollen is placed in corbiculae situated on the tibia of the bees hind legs to form the pollen loads, commonly known as the bee pollen, in the form of granules . Pollen S. aureus, Streptococcus spp, E. coli and P. aeruginosa with a higher sensitivity in Gram-positive bacteria [Bacillus cereus, S. aureus, S. typhi and E. coli were sensitive to Portugal bee pollen [B. cereus, B. subtilis, E. coli, Salmonella typhimurium, S. aureus, Yersinia enterocolitica, E. faecalis and Listeria monocytogenes did not exert any antimicrobial effect on these bacterial strains indicating that the activity is concentration dependent [Clostridium butyricum, Clostridium hystoliticum, Clostridium intestinale, Clostridium perfringens and Clostridium ramosum. Results showed an antibacterial activity against all of the above mentioned strains of clostridia with that against C. butyricum and C. perfringens being the most potent one [E. coli, P. aeruginosa, S. aureus and S. pyogenes showed a high sensitivity to the bioactivity of bee pollen in opposition to E. coli and P. aeruginosa [Bee pollen is often collected by using a pollen trap at the entrance or in the hives. Sometimes, it is directly collected from the bee hind legs . Water, bacteria . Similare pollen . Applyinependent . The anttent one . The antruginosa .L. monocytogenes, P. aeruginosa, S. aureus, S. enterica and E. coli. Results showed that S. aureus was the most sensitive bacteria to the ethanolic bee pollen extract while S. enterica was the most sensitive one to the methanolic extract [E. coli, S. enteritidis, S. aureus, L. monocytogenes were tested with ethanol pollen extract. It was found that S. aureus and L. monocytogenes growth was inhibited while this was not the case for both E. coli and Salmonella enteritidis which both showed resistance to ethanol extracts of pollen [E. coli and Campylobacter jejuni) than Gram-positive bacteria (L. monocytogenes) [S. pyogenes only and showed no activity against E. coli, S. aureus and P. aeruginosa [E. coli was the most sensitive strain to 70% ethanol bee pollen extract but resistant to 96% ethanol extract which confirms the fact that bacterial strains are specific to the solvent and its concentration [Furthermore, ethanol and methanol extracts of Slovakian bee pollen were tested for their antibacterial property against  extract . E. colif pollen . In sometogenes) . The samruginosa . Moreoventration .The antibacterial mechanism of bee pollen remains unclear until now. Studies suggest that the antibacterial activity of bee pollen is associated with glucose oxidase; an enzyme produced by honeybees and added to pollen during the process of granules formation . AdditioRoyal jelly (RJ), a yellowish white, sticky acidic secretion, is produced by worker bees and secreted from hypopharyngeal and mandibular glands. Bee larvae at early stages of their life rely on royal jelly as a primary source of food. Only the queen bee is fed by royal jelly until it dies, that’s why royal jelly is widely recognized as a ‘’super food’’ ,159.Water is the major molecule forming RJ (50 to 60%). In addition to water, RJ contains other components such as proteins (18%), carbohydrates (15%), lipids (3 to 6%), mineral salts (1.5%), and other minor constituents such as free amino acids, vitamins (particularly vitamin B) and phenols such as flavonoids .2), jelleine-II (TPFKLSLHL-NH2), jelleine-III (EPFKLSLHL-NH2), and jelleine-IV (TPFKLSLH-NH2). Jelleine I, II, and III are derived from MRJP1 cleavage product and are responsible for the antibacterial activity of MRJP1 [S. aureus, S. saprophyticus and B. subtilis) and Gram-negative bacteria . The results revealed that jelleines-I and II had a broad-spectrum activity, while jelleine-III was less active, and jelleine-IV had no antimicrobial activity [P. larvae, B. subtilis and E. coli. This antibacterial activity of MRJP2 is attributable to its high mannose carbohydrate levels and its complex-type antennary carbohydrate structures [RJ exhibits important antibacterial activity. This antibacterial potential of RJ is due to the existence of some main bioactive constituents. Proteins play an important role in the antibacterial activity of RJ. In fact, proteins are the major constituents of the dry matter of RJ (50% of the dry matter of RJ) ,163,164.of MRJP1 ,169. A pactivity . Hence, ructures . In addiructures . It is iructures .P. larvae, the main pathogen of American foulbrood disease. Royalisin also acts on other Gram-positive bacteria like Staphylococcus, Streptococcus, B. subtilis, Sarcina lutea, Micrococcus luteus and Gram-positive rods such as Corynebacterium, Clostridium, Lactobacilus helveticus and Leuconostoc. However, no inhibition of Gram-negative bacteria like E. coli and Serratia marcescens was detected [Royalisin, another antibacterial protein found in RJ, prevents RJ from becoing contaminated and infected by Gram-positive bacteria. Royalisin has been shown to have antibacterial activity against Gram-positive bacteria but not Gram-negative bacteria . Howeverdetected ,174,175.A study was conducted with the aim to assess the amount of royalisin in RJ obtained from honeybee colonies with different genotypes. It was found that there is an obvious link between the amount of royalisin and the antibacterial potential of RJ. Furthermore, the amount of royalisin in RJ was discovered to differ between colonies .E. coli) showed that royalisin inhibits bacterial growth by disrupting the permeability of the cell membrane as well as reducing the hydrophobicity of bacterial cells [The mechanism of action of the majority of RJ peptides remains unrecognized. For the first time, the analysis of RAcc-Royalisin  fatty acid, is the most abundant component in the lipids fraction (80%) and can only be found in RJ [E. coli) and Gram-positive  [Streptomyces species, S. aureus and E. coli. The results show that at concentrations of 30 mg/mL, ether-soluble fractions of RJ strongly reduced the growth of the previously described microorganisms, while non-soluble fractions had almost no antimicrobial activity [S. mutans colonization and pathogenesis. Yousefi et al. discovered that 10-HDA inhibits gene expression and mRNA transcription while also effectively reducing S. mutans adhesion to the cell surface [Moreover, nd in RJ . Unlike nd in RJ . Thus, tnd in RJ . Early s aureus) . Also, aactivity . Further surface .S. mutans and Streptococcus viridans, as well as S. aureus and S. epidermidis, were efficiently inhibited by 3-hydroxydodecanedioic acid, 10-acetoxy-2-decenoic acid, and (11S)-hydroxydodecanoic acid. However, most of these fatty acids occurred to be ineffective against Gram-negative bacteria. Moreover, 3-hydroxydodecanedioic acid, with a MIC of 0.47 mg/mL, appeared to have a minor effect on K. pneumonia [Milliou and Chinou reported other fatty acids that displayed an antibacterial activity. Oral pathogens neumonia .Consequently, some of MRJPs, jelleines, royalisin and 10-HAD may act synergically and confer the RJ an effective and promising antibacterial activity.K. pneumoniae which was not inhibited by neither samples. Sample A also had no effect on Streptococcus uberis [M. luteus, B. cereus and S. aureus) and Gram-negative bacteria . Results demonstrated that RJ exhibited an antibacterial activity and had two distinct forms of effect, one being bactericidal and the other being bacteriostatic against the studied bacteria. Likewise, they investigated the impact of storage time on RJ antibacterial activity. Within 24 h of being frozen, the highest antibacterial activity of RJ was observed. This activity decreased gradually over time until it reached a steady value [Raw RJ has been tested against human pathogens in many studies. Two RJ samples, from Argentina, A and B were tested against Gram-positive and Gram-negative bacteria that cause infections in cutaneous wounds. Both samples had the ability to inhibit Gram-positive and Gram-negative bacteria except for s uberis . Differes uberis . Anotherdy value . A recendy value .S. aureus and E. coli was 1.7% (v/v) and 2% (v/v), respectively. When mixed with starch, the MIC decreased by 61% and 30% against S. aureus and E. coli, respectively. It was suggested that this decrease in MIC values is due to the existence of amylases in RJ which might be a possible explanation for the increase in the antibacterial activity after adding starch. This enzyme breaks down starch into dextrin and maltose, increasing RJ osmotic capacity and, as a consequence, its antibacterial impact [P. aeruginosa [The combination of RJ with other compounds was also studied in order to enhance the effectiveness of RJ antibacterial activity. Boukraa et al. investigated the effect of mixing RJ with starch. They found that starch alone does not have an antibacterial activity. RJ MIC against l impact . Surprisruginosa .S. aureus and L. monocytogenes [Furthermore, Romanelli et al. found that when jelleine-I was combined with temporin A or temporin B (peptides from the temporin family that are active against Gram-positive bacteria at low concentrations), the antibacterial activity of jelleine-I was increased against ytogenes .The use of beehive products for medicinal purposes has long been exploited by mankind. Here, the antibacterial properties of honey, bee venom, propolis, pollen and royal jelly have been discussed. Each of these products is characterized by the presence of bioactive compounds which makes the formers powerful inhibitors of pathogenic bacterial strains.


Scorpiothyrsus erythrotrichus belongs to Melastomataceae. Here, we present its complete plastome. To our knowledge, this is the first reported complete chloroplast genome of S. erythrotrichus. The complete plastome of S. erythrotrichus is 160,731 bp in length with a typical quadripartite structure, consisting of four regions: large single-copy (LSC) region , small single-copy (SSC) region , and two inverted repeat regions . It contains 128 genes . The overall GC content is 36.9% and in the LSC, SSC, and IR regions are 34.70%, 30.40%, and 42.50%, respectively. Our study contributes to the molecular phylogenetic studies of Scorpiothyrsus and Melastomataceae. Scorpiothyrsus erythrotrichus (Merrill & Chun) H. L. Li, a shrublets with sparsely hispid, occurs in sparse to dense forests, mountain slopes, shaded places with an altitude of 600–1400 m, it is an endemic species to Hainan and included in threatened species list of China’s higher plants as critically endangered categories. Due to its spotted leaves, S. erythrotrichus has the potential for horticultural applications. With the rapid changes in climate and intensification of human activities, the distribution of S. erythrotrichus is rapidly reduced in recent decades. Understanding the spatial genetic pattern and demographic dynamics of the species can provide important guidelines for the protection and utilization of endemic species. Scorpiothyrsus is originally described as part of Phyllagathis . Fresh leaves were put into silica gel to preserve until DNA extraction. The voucher specimens were deposited in the herbarium of Sanya University (collector and collection number: Lang-xing Yuan (Langxingyuan@sanyau.edu.cn), BLHMJHD1). Genomic DNA of S. erythrotrichus was extracted according to CTAB method (Doyle and Doyle S. oligotrichus (MK994794) as a reference, then coupled with manual adjustment using Geneious v.10.1.3 .The sample of S. erythrotrichus was a circular molecule of 160,731 bp in length. It had a typical quadripartite structure including one large single-copy (LSC) region , one small single-copy (SSC) region , and two copies of inverted repeat regions (IRs) . The overall GC content was 36.90%, while the corresponding values of the LSC, SSC, and IR regions were 34.70%, 30.40%, and 42.50%, respectively. A total of 128 genes were encoded, of which 113 were unique and 17 were duplicated in the IR regions. Among the unique genes, 79 were protein-coding genes, 30 were tRNA genes, and four were rRNA genes.The complete plastid genome of S. erythrotrichus across the Melastomataceae, complete plastid genomes of S. erythrotrichus and other 12 species of Phyllagathis and S. oligotrichus (MK994794) were selected to conduct analyses, using Tigridiopalma magnifica (NC_036021.1) as outgroups  and Calanthe davidii (GenBank accession no. NC_037438). Filtered reads were then assembled into contigs in the software Platanus version 1.2.4. The physical map of the new chloroplast genome was generated using OGDRAW. Finally, the validated complete cp genome sequence was submitted to GenBank with accession number MN577470.The total genomic DNA was extracted from fresh leaves according to the methods described by Doyle and Doyle  and sequAnnamocarya sinensis were collected from Xichou county of Yunnan province. Total genome DNA was extracted with the Ezup plant genomic DNA prep kit . The voucher specimens of A. sinensis were deposited at the herbarium of Southwest Forestry University (accession number: SWFU-YAB-H-0160), and DNA samples were properly stored at Key Laboratory of State Forestry Administration on Biodiversity Conservation in Southwest China, Southwest Forestry University, Kunming, China. Total DNA was used to generate libraries with an average insert size of 350 bp, which were sequenced using the Illumina HiSeq X platform. Approximately, 14.0 GB of raw data were generated with 150 bp paired-end read lengths. Then, the raw data were used to assemble the complete cp genome using GetOrganelle software with Juglans nigra as the reference. Genome annotation was performed with the program Geneious R8  by comparing the sequences with the cp genome of Juglans nigra. The tRNA genes were further confirmed through online tRNAscan-SE web servers. A gene map of the annotated A. sinensis cp genome was drawn by OGdraw online.The fresh leaves of DNA with good integrity and purity was used for library construction and sequencing with the Illumina Hiseq-2500 .


A higher intake of dietary fiber is associated with a decreased risk of chronic inflammatory diseases such as cardiovascular disease and inflammatory bowel disease. This may function in part due to abrogation of chronic systemic inflammation induced by factors such as dysbiotic gut communities. Data regarding the detailed influences of long-term and recent intake of differing dietary fiber sources on the human gut microbiome are lacking.In a cohort of 307 generally healthy men, we examined gut microbiomes, profiled by shotgun metagenomic and metatranscriptomic sequencing, and long-term and recent dietary fiber intake in relation to plasma levels of C-reactive protein (CRP), an established biomarker for chronic inflammation. Data were analyzed using multivariate linear mixed models.Prevotella copri carriage in the gut, whereas those with P. copri carriage maintained stable CRP levels regardless of fiber intake.We found that inflammation-associated gut microbial configurations corresponded with higher CRP levels. A greater intake of dietary fiber was associated with shifts in gut microbiome composition, particularly Clostridiales, and their potential for carbohydrate utilization via polysaccharide degradation. This was particularly true for fruit fiber sources . Most striking, fiber intake was associated with significantly greater CRP reduction in individuals without substantial Our findings offer human evidence supporting a fiber-gut microbiota interaction, as well as a potential specific mechanism by which gut-mediated systemic inflammation may be mitigated.The online version contains supplementary material available at 10.1186/s13073-021-00921-y. Chronic low-grade systemic inflammation has been associated with several diseases that collectively comprise the leading causes of death worldwide, including cardiovascular disease, cancer, diabetes mellitus, and chronic kidney disease . EmerginEubacterium eligens [Numerous epidemiological studies have linked higher dietary fiber intake to reduced risk for several chronic inflammatory diseases , with th eligens . HoweverBifidobacterium or fiber could prevent these mucus aberrations [Bifidobacteria and increased fecal concentrations of beneficial SCFAs, such as butyrate [Independent of health outcomes, decreased dietary fiber intake has also been associated with gut microbial responses. These include overall lower gut microbial diversity , specifirrations . In healbutyrate , 18.Prevotella copri carriage.We thus performed a population-based study to examine the relationship between dietary fiber intake, the gut microbiome, and chronic systemic inflammation. This employed a subcohort of 307 men, the Men’s Lifestyle Validation Study (MLVS), nested within the Health Professionals Follow-Up Study (HPFS). We assessed stool metagenomes and metatranscriptomes in association with long-term and recent dietary fiber intake from various food sources. This served to elucidate the mechanisms underlying the direct effects of fiber intake on the microbiome and their potential direct and indirect effects on circulating C-reactive protein (CRP), a validated and widely accepted marker of systemic inflammation. Mildly elevated CRP levels have been associated with risk of cardiovascular disease and other chronic inflammatory diseases among initially healthy population –21. We fThe Health Professionals Follow-Up Study (HPFS) is an ongoing prospective cohort study of 51,529 US male health professionals aged 40 to 75 years at enrollment in 1986 . ParticiFrom 2012 to 2013, we recruited 307 men in the MLVS and collected longitudinal stool samples . ParticiAs previously described , we usedIn the HPFS, dietary intake was assessed every 4 years since enrollment in 1986 with a validated, semi-quantitative Food Frequency Questionnaire (FFQ) . ParticiParticipants in the MLVS were also administered 7-day dietary records recording recent diet contemporaneously with stool sample collections. Participants measured and reported gram weights for foods using a Primo Multifunction Kitchen Scale and ruler before and after eating, provided recipes of home-prepared foods, and returned labels of store-brand products. The Nutrition Data System for Research was used to derive over 150 nutrients and dietary constituents including dietary fiber intake .http://huttenhower.sph.harvard.edu/kneaddata) to remove low-quality read bases and reads of human origin. Taxonomic profiling was performed using MetaPhIAn 2.6.0 (http://huttenhower.sph.harvard.edu/metaphlan2) [Taxonomic and functional profiles were generated by using the bioBakery meta’omics workflow . Sequencaphlan2) , which chttp://huttenhower.sph.harvard.edu/humann) [Metagenomes and metatranscriptomes were functionally profiled using HUMAnN 2.11.0 (/humann) . Briefly/humann) . Unmappe/humann)  using DI/humann) . Hits ar/humann) , and (3)/humann) . More de/humann) , 25.Metatranscriptomic functional activity was assessed in 372 samples with both RNA and DNA data using RNA/DNA ratios. Owing to the compositionality of RNA and DNA measurements, the resulting ratio is relative to the mean transcript abundance of the entire microbial community. Thus, a ratio of 1 implies that the pathway is transcribed at the mean transcription abundance of all pathways in the microbial community. Infinite values of RNA/DNA ratios were imputed using 99% percentile of a given feature and values of 0 were imputed using half of the 1% percentile before log2 transformation. We excluded metagenomes and metatranscriptomes that did not surpass a minimum prevalence (10% for DNA and 20% for RNA/DNA ratio) and relative abundance (0.001% for DNA) threshold.The MLVS collected two fasting blood samples in blood tubes with liquid sodium heparin during the same period as fecal sample collection. After collection, tubes were placed in styrofoam containers with ice packs, returned to the laboratory via overnight courier, and centrifuged and aliquoted for storage in liquid nitrogen freezers (−130°C or colder). Plasma levels of high-sensitivity CRP were measured at the Franke lab at University of Hawaii using a Cobas MiraPlus clinical chemistry analyzer  and a latex particle enhanced immunoturbidimetry-based kit from Pointe Scientific . We excluded samples with high-sensitivity CRP levels below (<0.01 mg/L) or above (>20 mg/dL), the detection limits.We paired longitudinal stool microbiome with concurrent blood measurements and dietary intake, resulting in 925 samples for taxonomic and metagenomic analyses and 372 samples for metatranscriptomic analyses. We used the Bray-Curtis dissimilarity metric to determine the differences of the taxonomic composition at the species level, functional potential (DNA), and functional activity (RNA/DNA ratio). We performed omnibus testing with permutational multivariate analysis of variance (PERMANOVA) of Bray-Curtis dissimilarities (999 permutations) to quantify the percentage of variance explained by age, lifestyle, diet, and clinical biomarkers. We identified microbial species and functions associated with dietary fiber intake and CRP using multivariate linear mixed model in MaAsLin 2 0.99.12 . All modP. copri (fiber: P. copri) in addition to their main effects and evaluated the statistical significance of the product term:We additionally adjusted for body mass index (BMI) in the model for CRP to delineate microbial species associated with chronic inflammation independent of adiposity. To examine an interaction between fiber intake and gut microbiome composition, we applied an equivalent multivariate linear mixed model that included a product term of fiber and presence of p value of <0.25 was considered as statistically significant, with some results limited to a subset for visualization and full results in the supplement.Overall type I error was controlled using the Benjamini-Hochberg false discovery rate (FDR). A corrected r =−0.24), but not correlated with age (r =−0.02)  Fig. C. The so02) Fig. D. The ma02) Fig. .Fig. 1LWe included 925 metagenomes and 372 metatranscriptomes in our analyses . A total2<0.01; Additional file 2=0.0095, p = 0.005) and functional potential . Thus, neither fiber intake nor CRP levels alone were the main drivers of overall microbiome configurations, which were instead dominated by baseline inter-individual differences [We first tested for associations between overall microbiome structure and our main variables of interest (fiber subsets and CRP) and covariates  did not appear to maintain the expected protective relationship between increased fiber and lower CRP. Consistent with other findings [P. copri. We tested this interaction quantitatively and found that the inverse association between dietary fiber and CRP was significantly stronger among participants who did not have P. copri, compared to those with P. copri carriage , we observed that individuals in the outgroup of high findings , 24.1% oP. copri carriage, likely modifies the effects of fiber intake in alleviating chronic inflammation. Relatedly, P. copri has previously shown both positive and negative influences on human health. Some studies have linked P. copri to improved glucose tolerance and insulin responses in fiber-rich diets [P. copri with insulin resistance and glucose intolerance as well as inflammatory diseases [P. copri [These results support that microbiome structure, primarily via ch diets , 41. Othdiseases –44. In cP. copri  and the Eubacterium eligens, Faecalibacterium prausnitzii, and genus Roseburia, but also decreases in Clostridium, Lachnospiraceae, and Ruminococcus spp. Increased fiber intake was also associated with increased relative abundances of Haemophilus parainfluenzae and Bacteroides cellulosilyticus. These associations remained robust despite additional adjustment for Bristol score and other lifestyle factors including alcohol intake and physical activity.In the absence of overall microbiome shifts with fiber intake or CRP, we next identified individual microbial species associated with these variables using multivariate linear mixed model in MaAsLin 2  Fig. 3)3). All mE. eligens and F. prausnitzii and inverse associations with Lachnospiraceae and Ruminococcus were largely driven by pectin and fruit fiber. The distinct chemical structures of dietary fibers lead to substantial variations in solubility and fermentability and subsequent effects on the microbial composition and functions [F. prausnitzii and a highly selective promotion of E. eligens as well as depletion of Bacteroidetes [Greater microbial differences were observed in association with intake of pectin and fiber from fruits and, to a lesser extent, cereals, compared to vegetable fiber. For example, the findings mentioned above including positive associations with unctions , 10. Pecunctions . Our resroidetes –50. Assoroidetes .B. uniformis, B. salyersale, Barnesiella intestinihominis, and Parabacteroides independent of adiposity. In a previous analysis of 178 older adult subjects, CRP was positively associated with a metagenomically assembled Bacteroides co-abundance group [P. distasonis and P. johnsonii, the former was shown to alleviate obesity and metabolic dysfunctions via production of succinate and secondary bile acids in mice [Lachnospiraceae bacterium 3 1 46FAA, E. eligens, and Bifidobacterium bifidum, consistent with their anti-inflammatory effects as shown in experimental studies [In multivariate models for CRP, higher CRP was associated with enrichment of ce group . However in mice . Higher  studies , 54, 55.d-glucan linkages in cellulose) and EC 2.4.1.1 (glycogen phosphorylase), both of which play a role in catalyzing the degradation of glycans or polysaccharides.Dietary fiber intake in particular recent intake from pectin was also significantly associated with a large number of metagenomic functional pathways  . CAZy cod-galacturonic acid, and the fermentation requirement of pectinase, we detected an enrichment of PL9 strongly positively correlated with pectin intake. PL9 covers enzymes including pectate lyase (EC 4.2.2.2), exopolygalacturonate lyase (EC 4.2.2.9), thiopeptidoglycan lyase (EC 4.2.2.-), and rhamnogalacturonan endolyase (EC 4.2.2.23) and participates in the degradation of homogalacturonan. In our samples, expression of PL9 was primarily contributed by E. eligens, followed by B. thetaiotaomicron, F. prausnitzii, and B. sp. 1_1_6  modifies a dietary exposure (fiber intake) with a well-established marker of systemic inflammation (plasma C-reactive protein levels). In addition to this interaction effect, direct effects of dietary fiber intake on the gut microbiome generally increased Clostridiales, which also play a pivotal role in regulating localized and systemic inflammation [E. eligens and F. prausnitzii as well as their functions in the degradation of polysaccharides were enriched in participants with greater dietary fiber and, especially, pectin intake. We also linked individual microbial signatures to chronic systemic inflammation. These findings collectively offer novel human evidence supporting a variety of fiber-gut-microbiome interactions relevant to chronic systemic inflammation.Here, we have demonstrated one of the first explicit interaction relationships by which a specific component of the gut microbiome  [P. copri has been associated with chronic inflammatory conditions such as rheumatoid arthritis [P. copri complex may contribute to its functional diversity and some of these apparent phenotypic contradictions [P. copri isolates utilize distinct sets of polysaccharides from dietary plant sources [P. copri diversity, fiber type and amount diversity, and the gradual, multi-generational loss of P. copri clades from Westernized populations could account for the complexity of this interaction [P. copri could in principle have both direct effects on systemic inflammation, as well as opposing, indirect effects caused by reduced bioavailability of fermentable fibers or other fermentation products to other microbes. Additional investigation is thus needed to functionally characterize the influence of P. copri on modulating dietary effects on inflammation, health, and host-microbe coevolution.Specifically, in our population, pionate) , 58 and pionate) , 41. Conrthritis , 43 and rthritis . Strain-dictions , 60. For sources . The comeraction . Our finB. thetaiotaomicron [F. prausnitzii [E. eligens [An additional intriguing result from this study was the specificity of many fiber-microbiome influences to fruit fibers and pectin. As a major soluble fiber component in the plant cell wall, particularly in fruits and vegetables, pectin serves as the nutritional niche for some groups of bacteria, such as aomicron , F. prauusnitzii , and E.  eligens . It is l eligens . Polysac eligens . However eligens .At least one additional recent study, using a distinct population and methodology, found potentially similar between-subject variation in fiber sources with respect to the microbiome. There, significant agreement between microbiome composition and fiber-source diversity was observed for fruits and grains, but not for vegetables or legumes . Such heP. copri mediates dietary biochemistry and host inflammation, as well as the specific routes by which pectin directly influences other gut microbiome members. An understanding of these distinct effects of dietary fibers, pectin, and how they are transformed and utilized by microbial communities would pave the way forward for development of personalized fiber-based interventions for the prevention of chronic inflammatory diseases.As one of the only sustainable long-term influences on the gut microbiome and chronic health, dietary interactions and interventions are a key strategy to mitigate chronic inflammation. Our findings will benefit from further investigation of the specific mechanisms by which Additional file 1: Figures S1-S6.Additional file 2: Tables S1-S6.


Leucostethus from Gorgona Island, a small (13 km2) island located 35 km from the Pacific coast of southern Colombia. The new species most resembles L.argyrogaster and L.fugax from western Amazonia at 340–870 m elev. in Peru and Ecuador, with which it shares pale ventral coloration and orange suffusion of the axilla, groin and concealed surfaces of the hind limb, but it is most closely related to L.bilsa from ca. 340 km SW in the southern Chocó at 420–515 m elev., northwestern Ecuador. We report miniscule white spots on the posteroventral surface of the thighs of the new species and, on the basis of our preliminary assessment of their taxonomic distribution, hypothesize that their presence is a synapomorphy of Dendrobatoidea with subsequent losses in a few groups. Given the apparent restriction of the new species to Gorgona Island, it is Vulnerable according to IUCN Red List criteria. In addition to naming the new species, we also propose the following new combinations: L.alacris  comb. nov., L.dysprosium  comb. nov., and L.yaguara  comb. nov.We describe a new species of  See CPZ-UV). We also include in the type series one specimen from the National Museum of Natural History, Smithsonian Institution, Washington DC, USA (USNM).We conducted fieldwork on Gorgona Island (PCM) recorder and internal microphone at a sampling rate of 44.1 kHz with 16 bit encoding at 26.2°C ambient temperature. We also captured audio and video of the holotype vocalizing using a Canon EOS Rebel T3 and internal microphone and analyzed the audio . The calling males were observed directly and recorded at ca. 1 m distance. Acoustic analyses employed standard definitions and terminology for spectral and temporal variables  emitted in bouts of < 30 s duration, we classified the individual units of sound as notes grouped into calls. This treatment is consistent with the interpretation by Colostethusfraterdanieli group, Leucostethus, and we follow their taxonomy here. Character definitions follow Anura and number fingers accordingly. As such, we follow Information on phylogenetic relationships was taken from SVL snout–vent lengthFAL forearm length from proximal edge of palmar tubercle to outer edge of flexed elbowHL hand length from proximal edge of palmar tubercle to tip of finger IVTL tibia length from outer edges of flexed knee to heelFL foot length from proximal edge of outer metatarsal tubercle to tip of toe IVHW head width between angle of jawsHLD head length diagonally from corner of mouth to tip of snoutEL eye length from posterior to anterior cornerEND eye-naris distance from anterior corner of eye to center of narisIND internarial distance between centers of naresSL snout length from anterior corner of eye to tip of snoutIOD interorbital distanceTD tympanum diameterx̄ ± SE. Males with vocal slits on both sides of the mouth were scored as adults, those with only one vocal slit as sub-adults, and those lacking slits on both sides as juveniles. Females with expanded, convoluted oviducts and enlarged oocytes were considered to be adults, those with only weakly expanded, non- or weakly convoluted oviducts and poorly differentiated oocytes to be sub-adults, and those with small, undifferentiated oocytes and unexpanded, straight oviducts to be juveniles. Color in life is based on field notes and digital photographs.Unless otherwise noted, measurements and proportions are given only for adults and are summarized as Leucostethus sensu L.argyrogaster ; L.bilsa, L.brachistriatus , L.fraterdanieli , L.fugax , L.jotaL.ramirezi ], to which we add L.alacris  comb. nov., L.dysprosium  comb. nov., and L.yaguara  comb. nov. Although the precise phylogenetic relationships of these three species have not yet been tested in quantitative phylogenetic analyses, L.alacris and L.yaguara to L.fraterdanieli and L.fraterdanieli might correspond to one or both of those names. Similarly, L.dysprosium shares a complete, continuous, pale oblique lateral stripe with other species of Leucostethus (see Discussion) and appears to differ from other trans-Andean species of Leucostethus except L.alacris only in possessing more extensive toe webbing . We also compare the new species to similar colostethines from the Chocó.We compare the new species to other species of Taxon classificationAnimaliaAnuraDendrobatidaeGrant & Bolívar-Garcíasp. nov.7857DA04-3F6E-5A7F-A73F-C498ACF66AC0http://zoobank.org/757C0F3D-D62C-40E2-8F77-195C9234842DColostethus sp. Gorgona: Grant et al. (2017)Leucostethus sp. Gorgona: Vigle et al. (2020)Holotype.CPZ-UV 7293 (field number WB 3045), an adult male collected by Taran Grant immediately west of the housing complex at El Poblado, Gorgona Island, Parque Nacional Natural Gorgona, Guapi, Cauca Department, Colombia, 02°58'00.6"N, 78°10'27.4"W, ca. 30 m elevation, 26 May 2016.Paratypes.CPZ-UV 5013–5014, CPZ-UV 7294–7297, collected at the type locality by Wilmar Bolívar-García, Taran Grant, David Andrés Velásquez-Trujillo, and Andrés Felipe Gómez Fernández, 26–27 May 2016. USNM 313893, collected by Humberto Granados Diaz, Gorgona Island, 1987.Leucostethus  with complete pale oblique and ventrolateral stripes, dorsolateral stripe absent, finger IV of adult males very weakly swollen (barely discernible), throat of adult males bearing, at most, faintly stippled spotting or reticulation on throat, pale paracloacal spots present, axilla, groin, and concealed surfaces of hind limb suffused with orange, and toes II–IV with basal webbing.A moderate-sized Leucostethussiapida sp. nov. differs from all congeners except the cis-Andean species L.argyrogaster and L.fugax in possessing orange axilla, groin, and concealed surfaces of hind limb (yellow in other species) and lacking conspicuous dark coloration on the throat of adult males (present in other species); it differs from both species in being larger  and further differs from L.fugax in lacking conspicuous swelling of finger IV in adult males (strongly swollen in L.fugax).Leucostethus, L.siapida sp. nov. differs from all except L.bilsa (apparently unswollen) and L.alacris (unknown) in lacking conspicuous swelling of finger IV in adult . It differs from L.bilsa in the coloration of the axilla, groin, and concealed surfaces of the hind limb , male throat coloration , and female SVL  of L.siapida sp. nov. is more than twice that of the other species . The advertisement call of L.siapida sp. nov. also has a higher pitched dominant frequency than all species except L.jota .Among species of rdanieli , L.frated L.jota , and L.b and lacking a conspicuously swollen finger IV (conspicuously swollen in both species). It further differs from S.dalyi in lacking a discrete, dark brown postrictal spot and from S.nubicola in bearing, at most, faintly stippled spotting or reticulation on the throat .Among other anurans in the Chocó, l stripe . It diff(in mm).CPZ-UV 7293 is an adult male ; vocal slits present; swelling of finger IV barely discernible , mature oocytes creamy yellow and pale brown, ca. 1.8 mm diameter; oviducts enlarged, convoluted, creamy white. Large intestine unpigmented.The following description is based on the five adult males and three adult females in the type series; measurements are reported in Table Ventral and most dorsal surfaces smooth; exposed surface of shank with low, inconspicuous granules. Postrictal and cloacal tubercles absent.SVL and 1.0–1.2 times diagonal HL in males, 32–34% and 1.0–1.1 in females, IOD 29–36% of HW. Snout bluntly rounded in dorsal view, SL 46–58% of HL. Nares slightly flared, directed posterodorsad, EN 70–86% of EL and 57–66% of SL. Loreal region weakly concave, almost vertical. Canthus rostralis well defined, sharply rounded. Incomplete tympanic ring discernible externally along anteroventral half of tympanum. Eye length 38–48% of head length. Tympanum directed posterodorsad, TD 41–50% of EL in males, 52–53% in females. Supratympanic bulge associated with the underlying depressor musculature present. Teeth on maxillary arch present; median lingual process absent. Posterodorsal portion of tympanum concealed by m. depressor mandibulae fibers extending ventrad from origin on dorsal fasciae. Trigeminal nerve (V3) lateral to undivided m. levator mandibulae externus.Head width 33–36% of SVL and 1.0–1.1 times FAL. Relative appressed finger lengths IV > II > III = V  surface of foot light brown with dark brown cross bands and blotches. Anterior surface of the thigh with prominent dark brown longitudinal stripe, delimited dorsally by narrow white line extending from groin and fading distally. Thigh ventrally immaculate. Most specimens with posterior thigh bearing elongate, tapered, pale sickle-shaped paracloacal stripe extending along proximal half to entire length of thigh, bordered dorsally by brown stripe extending along posterior shank and ventrally by solid or diffuse pale brown stripe; hes Fig. . VentralUSNM 313839 with entire dorsal surface of arm and fingers IV and V suffused with brown and irregular small dark brown spots. Anterior and posterior surfaces of upper arm with well-defined dark brown longitudinal stripes. Dark pigmentation on posterior surface extending distad to wrap around ventral and outer surface of elbow along posterior forearm and finger II. Anterior surfaces of forearm and dorsal surfaces of fingers II and III creamy white. Palmar surfaces pale brown; contact surfaces of tubercles creamy white.Dorsal surface of arm proximally pale, distally becoming pale brown with variably expressed dark-brown blotches to fingers IV and V, except USNM 313839 with diffuse pale spotting below oblique lateral stripe and darker oblique lateral stripe. Oblique lateral stripe continuous, except left stripe of CPZ-UV 7294 with single narrow break at mid-length. Ventrolateral stripe  indistinguishable from immaculate venter (e.g. CPZ-UV 5014) or evidenced solely by sparse melanophores along ventral edge (e.g. CPZ-UV 7294), extending anteriad above arm insertion, below tympanum, and below eye to tip of snout, on head delimited ventrally by suffusion of sparse melanophores terminating beneath naris . Dark brown of flank extending anteriad over tympanum to posterior edge of orbit and continuing over loreal region and around snout (encompassing nares) to form dark brown face mask.Flank solid dark brown, divided diagonally by well-defined creamy white oblique lateral stripe from groin to posterior corner of orbit , except Throat, chest, and belly creamy white, at most bearing faintly stippled spotting or reticulation on throat and along ventral edge of ventrolateral stripe.Dorsum brown with blackish-brown blotches Fig. . Dorsal CPZ-UV 7293; 22.7 mm SVL; Suppl. material CPZ-UV 7295 (19.9 mm SVL) at 15:45 h  for centuries or more and were almost certainly the first humans to encounter this species of frog.The specific epithet, Epipedobatesboulengeri on the previous days without rainfall, individuals of Leucostethussiapida sp. nov. were only observed after ca. 6 h of heavy rain between ca. 5:00 and 11:00 h on 26 May 2016.Individuals were active during the day in and on leaf litter, low vegetation, and low objects on the forest floor  and encapsulated by thick connective tissue beneath which the testis is pigmented, while the right testis appears to be absent. With the exception of their pigmentation, the testes of all other individuals are typical of this group . Although this finding rules out the lipophilic alkaloid-based chemical defense found in many dendrobatids , its precise phylogenetic relationships are unclear. Leucostethus, whose species occur from ca. 1000–2700 m elev. in the Andes and Cauca River valley. In contrast, Leucostethus species by L.siapida sp. nov. is sister to L.bilsa, which was recently described from ca. 340 km SW in the Reserva Biológica Bilsa at 420–515 m elev., northwestern Ecuador  that are far removed from L.siapida sp. nov.Among the species of Leucostethus; however, distinguishing between absence and weak swelling is notoriously difficult . As such, extreme caution is required when employing absence versus weak swelling to diagnose similar species. For this reason, we did not distinguish between the reported absence of swelling in L.argyrogaster and L.bilsa and the weak swelling in L.siapida sp. nov.Scoring the occurrence of swelling on finger IV of adult males is unproblematic in species that exhibit conspicuous swelling , as do most species of ifficult . For exaLeucostethussiapida sp. nov. and its sister species L.bilsa is the lateral variation in testis melanization. Although ontogenetic variation in testis pigmentation is common, individual variation among adults is rare, and, prior to L.bilsa  by A curious characteristic shared by  L.bilsa , unilateDendrobatoidea. Although an exhaustive review was beyond the scope of the current study, they also occur in additional species of Leucostethus  from type series or topotypical samples.” Nevertheless, it should not be overlooked that the persistence of these species in Colostethus for the past 15 years was not due to empirical evidence, but rather (1) their placement in Colostethus prior to its partitioning into multiple genera by Leucostethus. Although referral of these species to Leucostethus is not based on either quantitative phylogenetic analysis or clear synapomorphies and is, therefore, more predictive than explanatory, it is not entirely unsubstantiated by morphology. All three species share with other species of Leucostethus the presence of a complete (i.e. groin to eye), continuous, pale oblique lateral stripe (incomplete in some specimens of L.bilsa). In contrast, the oblique lateral stripe is either absent , or complete but formed by a series of spots .In addition to describing the new taxon Leucostethussiapida sp. nov. will be found on the mainland, based on current knowledge, L.siapida is endemic to Gorgona Island. Although Gorgona Island is protected as Parque Nacional Natural (PNN) Gorgona, the fact that the only known population is confined to an island of only ca. 13 km2 is sufficient to categorize this species as Vulnerable according to the IUCN Red List criteria (criterion D2; Despite our optimism that additional populations of


Skin colonization with coagulase-negative staphylococci (CoNS) is generally beneficial, but recent investigations suggest its association with flares and atopic dermatitis (AD) severity. However, this relationship remains unclear.To assess patterns of staphylococcal colonization and biofilm formation in toddlers with and without AD from rural and urban South African settings.in-vitro biofilm formation.We conducted a cross-sectional study of AD-affected and non-atopic AmaXhosa toddlers from rural Umtata and urban Cape Town, South Africa. CoNS isolates were recovered from lesional, nonlesional skin samples and the anterior nares of participants. Identification of the staphylococci was achieved by MALDI-TOF mass spectrometry. The microtiter plate assay assessed S. aureus commonly co-colonized nonlesional skin among cases , and anterior nares in urban cases  than the control group. S. capitis colonization on nonlesional skin and anterior nares was positively associated with more severe disease in rural  and urban cases , respectively. Biofilm formation was similar between cases and controls, independent of rural-urban living.CoNS and CoNS colonization is associated with AD and disease severity and may be implicated in AD exacerbations. Studies are needed to understand their underlying pathological contribution in AD pathogenesis. Staphylococcus represents the largest bacterial group on healthy skin and anterior nares ) are risk factors for CoNS colonization among urban toddlers  of controls had no staphylococcal colonization on nonlesional skin compared to the nonlesional skin of cases . Co-colonization with CoNS species and S. aureus on nonlesional skin was more prevalent in cases from both rural  and urban  settings than controls. Similarly, the colonization rates for only S. aureus or CoNS were higher on the nonlesional skin of cases than controls, independent of geographic location. Urban controls had no co-colonization with CoNS species and S. aureus in their anterior nares  vs. 0% [0/58], P = 0.029). Moreover, S. haemolyticus prevalence was similar among lesional cases in the rural (10%) and urban (11%) cohorts  (P = 0.009)  and anterior nares (4.3% [2/46] vs. 0% [0/34]) of cases compared to controls in the urban cohort  and anterior nares (P = 0.0004) in urban cases. This trend was also observed with S. capitis nonlesional skin colonization compared to anterior nares (P = 0.027) but not AD lesional skin (P = 0.069) among urban cases.s Tables . Also, S= 0.029) . However= 0.009) . Overalln cohort . CompariS. aureus and CoNS on lesional skin . This when was compared to those without staphylococcal colonization , and those by CoNS only  , and to those co-colonized with S. aureus and CoNS   and rural (76.5 [68–81.5] vs. 44 [P = 0.0059) settings (P = 0.031) .only  regardless of disease status, sampling site, or geographic location  of the staphylococci and its correlation with measures of diseases. The cross-sectional design prevents the assessment of the causality between staphylococcal colonization and biofilm phenotypes with AD severity. Although all cases had used emollients before sampling, we could not account for the timing of emollient administration before sampling. This scenario may have affected the patterns of staphylococcal colonization presently observed [The strengths of this study include the following. Firstly, a unique rural and urban cohort provided insights into how staphylococcal colonization differs between cases and controls in toddlers across geographies with distinct environmental exposures. Secondly, although observed  and may S. capitis, are associated with AD severity. This observation could play a role in the pathogenesis and exacerbation of AD through mechanisms not yet fully described in the literature. Furthermore, this may be dependent on their interactions with S. aureus. Our study highlights that while S. aureus remains the most studied Staphylococcus species in the pathogenesis of AD, CoNS may contribute to AD exacerbations. It also highlights the need for further in vivo studies on specific CoNS species or strains and how they contribute to AD exacerbation. Understanding the dynamics of the pathological role of CoNS will allow their targeting by therapeutic strategies aimed at countering complications from CoNS-associated infections in AD.In summary, although CoNS colonization is generally beneficial on healthy skin, we show that CoNS, particularly S1 Table(DOCX)Click here for additional data file.


Pseudomonas syringae has been recently identified against Caenorhabditis elegans. The current study was designed to investigate the detailed genetic mechanism of the bacterial pathogenicity by applying comparative genomics, transcriptomics, mutant library screening, and protein expression. Results showed that P. syringae strain MB03 could kill C. elegans in the liquid assay by gut colonization. The genome of P. syringae MB03 was sequenced and comparative analysis including multi locus sequence typing, and genome-to-genome distance placed MB03 in phylogroup II of P. syringae. Furthermore, comparative genomics of MB03 with nematicidal strains of Pseudomonas aeruginosa (PAO1 and PA14) predicted 115 potential virulence factors in MB03. However, genes for previously reported nematicidal metabolites, such as phenazine, pyochelin, and pyrrolnitrin, were found absent in the MB03 genome. Transcriptomics analysis showed that the growth phase of the pathogen considerably affected the expression of virulence factors, as genes for the flagellum, glutamate ABC transporter, phoP/phoQ, fleS/fleR, type VI secretion system, and serralysin were highly up-regulated when stationary phase MB03 cells interacted with C. elegans. Additionally, screening of a transposon insertion mutant library led to the identification of other nematicidal genes such as acnA, gltP, oprD, and zapE. Finally, the nematicidal activity of selected proteins was confirmed by heterologous expression in Escherichia coli.Nematicidal potential of the common plant pathogen  Caenorhabditis elegans has been widely utilized to study host–pathogen interaction. Pathogenicity mechanisms of numerous bacterial species of Bacillus   . Moreove elegans . In contG medium  and in aG medium  was medincognita . These oP. syringae MB03 required for pathogenicity against C. elegans in the liquid killing assay. Comparative genomics, transcriptomics, and transposon insertion mutant library screening were performed to predict and identify key virulence factors. Further, the selected nematicidal genes were heterologously expressed in Escherichia coli to evaluate their toxicity. Collectively from these experiments, various species-common and strain-specific novel virulence factors belonging to P. syringae were identified.This study was designed to investigate the key virulence factors of the P. syringae MB03  strains were cultured at 37°C for routine growth. The C. elegans wild-type N2 (Bristol) was used as a model host in all bioassays. C. elegans N2 wildtype was obtained from Caenorhabditis Genetics Center College of Biological Sciences, University of Minnesota, United States. The worms were maintained on NGM medium having E. coli OP50 as food source at 20°C , 40 μl S medium, and 30–40 L4 worms.To express red fluorescent protein (RFP), the plasmid pMCh-23  was intrP. syringae MB03 was performed using Illumina technology. Quality trimming of 150-nucleotide (nt) paired-end reads was produced from a 500-bp genomic library . For de novo assembly of the genome, SOAPdenovo software was used. Open reading frame calling and annotation was performed by using Glimmer software .To increase the understanding of the virulence mechanism of this bacterial strain, whole-genome shotgun sequencing of software . Blastalsoftware . The anagyrB, gapA, fruK, pgi, rpoD, anB, and gltA was performed to further classify P. syringae MB03 as reported previously   using seven housekeeping genes including eviously . The P. of Mauve . GC skeww Server . To dist species . For thi > 0.64) . The EDG > 0.64) . In addi > 0.64) . Among tP. syringae MB03 genome. Previously, the nematicidal proteins of P. aeruginosa strain PA14 and strain PAO1 have been identified by screening the transposon insertion mutant libraries    and theiP. syringae MB03 were retrieved from the NCBI database. Physiochemical properties, including molecular weight, aliphatic index, stability, and isoelectric points of hypothetical proteins, were identified by ProtParam server  exponential phase and (II) stationary phase]. For this purpose, a single pure colony of P. syringae MB03 was cultured in LB broth. The overnight culture was used to further inoculate two different groups  which were incubated at 28°C with shaking at 160 rpm for 12 h. Bacterial cells were harvested by centrifugation and washed repeatedly with S buffer. Finally, pre-fasting L4 C. elegans were exposed to bacterial cells in NGM broth for 5 h. After a specified time of interaction, worms were removed by centrifugation and washed repeatedly to obtain bacterial cells. This resulted in the fabrication of the exponential phase group. The pellets of bacterial cells were used to extract total RNA (P. syringae MB03 exposed to C. elegans as treatment and P. syringae MB03 incubated without C. elegans as control). For the stationary phase group, the entire sample treatment was the same, except the bacterial cells were grown for 24 h so that the cells might enter the stationary phase of growth. After 24 h growth, bacterial cells were exposed to pre-fasting L4 worms for 5 h. Worms were removed after a specified time interval. The bacterial cell pellet was obtained by repeated washing of worms. A bacterial cell pallet was used to isolate total RNA from the stationary phase bacterial cells for transcriptomics (P. syringae MB03 exposed to C. elegans as treatment and P. syringae MB03 incubated without C. elegans as control).RNA sequencing of the P. syringae MB03 and with reference genomes including P. syringae B728a, P. syringae DC3000 using Bowtie 2  and recipient cells (P. syringae MB03) were grown overnight in LB at 37 and 28°C, respectively. One ml from each culture was subjected to centrifugation. Bacterial pellets of donor cells and recipient cells were washed and re-suspended in 1ml of LB. One hundred μl of each re-suspended culture was mixed and spread on LB agar plate. After 24 h of incubation, bacterial cells were scraped from the agar and suspended in 1 ml phosphate-buffered saline (PBS). Appropriate dilutions of the bacterial growth were spread on MG agar plate (to inhibit the growth of donor strains) supplemented with 50 mg/ml kanamycin (to select the plasmid transformed cells of P. syringae MB03) and incubated at 28°C for 48 h.For the construction of transposon insertion mutant library, donor P. syringae MB03 attenuated mutants, the liquid killing assay was conducted. Briefly, mutants of P. syringae MB03 were grown for 24 h at 28°C. The cells were washed and diluted in S medium supplemented with 5 μg/ml cholesterol. Cell density was adjusted at 0.6OD600 and 150 μL of cell suspension was added into the assay. Worms were synchronized and 30–50 L4 synchronized worms were added in the assay as described previously  comparative genomics, and (3) prediction based upon an online web tool, VirulentPred  lentPred . Selecteproteins . RecombiP. syringae MB03 could kill C. elegans in the liquid assay. The percent mortality increased with an increase in cell inoculum ; the opacity of the line is representing the degree of similarity (The GC skew of MB03 was generated using the CGView server. All the predicted ORFs of MB03 were compared with the reference strains (milarity . The genmilarity .P. syringae were considered harmless to C. elegans (P. syringae MB03 could kill C. elegans. This raised the possibility of the presence of strain-specific genes in MB03 strain which could involve in host infection. The distinction between the core and dispensable genomes provides better insight into the phenotypes and virulence mechanisms of a strain (P. syringae was determined (40% identity over 40% length), and the analysis resulted in the determination of 3397 genes as the core genome of P. syringae species (P. syringae MB03 genome was compared (using 40% identity over 40% length as selection criteria) with the previously determined core genome by P. syringae MB03 was compared with DC3000, CC1557, B728a, HS191, B64, and SM  contains most of the hop effectors of this strain (Psyr_1175 to Psyr_1239). When compared with MB03, integrase, transposase, hopX1, avrB3 and two hypothetical proteins were absent. Alternatively, several additional unique proteins were found in MB03. The rest of the island was similar between the two strains  contained many strain-specific genes. The gene VT47_00420 encodes a LasR transcriptional regulator that is absent in the genome of HS191, B728a, DC3000, B64, SM, and CC1557. According to the KEGG pathway and previous literature reports, the homologs of LasR transcriptional regulators have a well-defined role in bacterial pathogenicity  for short-chain fatty acid degradation; hisJ (VT47_18480), which is a part of an ABC transporter for histidine transport; and gltI (VT47_18645), which might be correlated to a shift in nutrient availability. Additionally, pntA (VT47_24115) and scoB (VT47_15125) were up-regulated, whereas scoP (V47_01435), queD (VT47_04670), rpsT (VT47_03545), and two hypothetical proteins  were down-regulated during host–pathogen interaction regardless of the bacterial growth phase  were only up-regulated in the exponential phase cells. Another important difference in the gene expression pattern was observed in the case of ABC transporters primarily related to amino acids. In bacterial two-component systems, phoP/phoQ genes were up-regulated when interaction was studied with exponentially growing bacterial cells. This system has been documented to trigger various bacterial virulence factor during environmental stress and host pathogen interaction  and amino acid uptake and metabolism  were up-regulated during the exponential phase interaction. It has been reported that most of hosts enforce amino acid starvation to the invading bacteria. In response, bacterial pathogens manipulate the host metabolism to overcome nutrient depletion  are among the major factors in its plant host determination  and hrcQa (VT47_05955) were up-regulated when stationary phase MB03 cells were exposed to the worms. Both of these proteins are among the core proteins of Type III secretion system and share significant similarities with flagellar proteins , the entire cluster was up-regulated during stationary phase host-pathogen interaction. Two Rhs element Vgr proteins  (VT47_23935 and VT47_23990) were also up-regulated.Among bacterial secretion systems, the Type VI secretion system plays an important role in the host-microbe interaction and virulence . Howeverrization . Althoug0 and T1 . Comparereported . Except 54 dependent (VT47_11275) within the cluster and another LysR family protein (VT47_11320) downstream of the cluster. Previously, a σ54 dependent transcriptional regulator was reported to play an important role in the regulation of the T6SS clusters . This revealed 87 and 30 homologs of PA14 and PAO1, respectively, in MB03 , isovaleryl-CoA dehydrogenase (VT47_11890), putative acyl-CoA carboxylase alpha chain (VT47_11875), acyl-CoA carboxyltransferase beta chain (VT47_11885), prpC (VT47_09990), and PrpB (VT47_09985). On the other hand, crfX (VT47_10030), (VT47_19750) and hypothetical protein (VT47_10035) were down regulated . Among these 30 homologs, one named kdpB was markedly up regulated.Virulence factors of different pathogens, such as osa PA14  and 68 ientified . To furt in MB03 . Among t in MB03  upon hosegulated . RegardiP. aeruginosa against C. elegans  homologs, which were previously demonstrated to be essential for full bacterial virulence of  elegans . The res elegans . Glutamanitrogen .P. aeruginosa  in the genome of P. syringae MB03. Genes for betaine transport were also up-regulated during the exponential phase interaction. Transcriptional factors related to the AraC family have been reported for carbon metabolism, bacterial pathogenicity, and stress response  of P. aeruginosa PA14. Recently, the ZapE protein has been reported for its role in cell division, and the protein is required for bacterial infection  showed attenuated virulence. The protein can interconvert citrate and isocitrate thereby facilitating a shift in different metabolic pathways, including the citrate cycle and glyoxylate cycle. Similarly, the mutation in the oprD (V47_06900) gene resulted in attenuated pathogenicity against C. elegans. The gene oprD encodes porin which forms a channel in the bacterial cell membrane. Different functions have been associated with membrane porins, including resistance against antimicrobials, nutrient uptake, and adhesion  was predicted to be involved in host–pathogen interaction by the VICMpred server. This was also supported by our experimental data from the transposon insertion mutant library screening. Moreover, this protein is predicted as a transcriptional regulator based on homology modeling by the HPIDB server. Similarly, uncharacterized transcriptional regulators which showed highly differential expression were selected to predict target genes  during host–pathogen interaction, approximately 70% of the genes were part of the core genome. The fraction of the core genome also outnumbered the auxiliary genome during the determination of nematicidal homologs of P. aeruginosa and from mutant library screening. It should be noted that approximately 66% of genes of MB03 contribute to the core genome, and the remaining 34% of the genome is comprised of auxiliary and strain-specific genes. Hence, it can be concluded that the distribution of virulence factors was not biased toward the core genome or auxiliary genome. The core genome of P. syringae, which was employed in comparative genomics, was determined by considering three diverse phylogroups of the P. syringae species  homolog was found in MB03, and no significant change in the expression of hdtS was observed. Hence, hdtS did not appear to play a significant role in the killing of C. elegans.Different mechanisms have been proposed by which bacterial species kill nfection . In the on model . Compara sensing , a mechaand hdtS . Similarand hdtS . HoweverC. elegans , genes related to locomotion (flagella proteins), and genes for nutrient acquisition (different metabolic proteins and catabolic enzymes).In summary, The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/LL: conceptualization, funding acquisition, project administration, resources, supervision, and writing – review and editing. MA: formal analysis, investigation, and writing – original draft. TG, XY, AB, ZW, XS, and NA: validation and visualization. All authors: contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pneumocystis pneumonia (PCP). We hypothesized that MPA exerts selective pressure on P. jirovecii microorganisms considering its in vitro antifungal activity on other fungi. Thus, we analysed impdh gene in P. jirovecii isolates from SOT recipients. P. jirovecii specimens from 26 patients diagnosed with PCP from 2010 to 2020 were retrospectively examined: 10 SOT recipients treated with MPA and 16 non-SOT patients without prior exposure to MPA. The P. jirovecii impdh gene was amplified and sequenced. Nucleotide sequences were aligned with the reference sequences retrieved from available P. jirovecii whole genomes. The deduced IMPDH protein sequences were aligned with available IMPDH proteins from Pneumocystis spp. and other fungal species known to be in vitro sensitive or resistant to MPA. A total of nine SNPs was identified. One SNP (G1020A) that results in an Ala261Thr substitution was identified in all SOT recipients and in none of the non-SOT patients. Considering that IMPDHs of other fungi, resistant to MPA, harbour Thr (or Ser) at the analogous position, the Ala261Thr mutation observed in MPA-treated patients was considered to represent the signature of P. jirovecii exposure to MPA. These results suggest that MPA may be involved in the selection of specific P. jirovecii strains that circulate in the SOT recipient population.Mycophenolic acid (MPA) targets the inosine 5′-monophosphate dehydrogenase (IMPDH) of human lymphocytes. It is widely used as an immunosuppressant to prevent rejection in solid organ transplant (SOT) recipients who, incidentally, are at risk for  Pneumocystis jirovecii (P. jirovecii) is a transmissible, uncultivable fungus responsible for severe pneumonia in immunosuppressed patients. Pneumocystis pneumonia (PCP) remains the most frequent AIDS defining infection in HIV-infected patients in developed countries. In France, PCP represents 30% of the annually reported 1200 AIDS cases . Si. SiP. jictively) ,23,24 (Fctively) ,23,24. MP. jirovecii IMPDH was found only in the 10 SOT recipients enrolled in the study, all of them being still subject to anti-rejection treatment with MPA at the date of PCP diagnosis. Nonetheless, putative resistance of P. jirovecii to MPA is probably not a risk factor for PCP occurrence. Indeed, the patients developed PCP because none of them were undergoing chemoprophylaxis when P. jirovecii infections occurred. In fact, the post-transplantation period ranged from 8 months to 216 months, whereas the duration of recommended prophylaxis is limited to 6–12 months after transplantation [P. jirovecii isolates from SOT patients may represent a signature of MPA exposure and the circulation and transmission of specific P. jirovecii strains in susceptible patient populations. This signature of MPA exposure was previously pointed out in environmental fungal species such as Aspergillus glaucus, Aspergillus clavatus, Aspergillus terreus, which encode IMPDHs harbouring Ser or Thr at the analogous position [Penicillium species that naturally produce MPA [Interestingly, the mutation Ala261Thr in antation . Howeverposition . These educe MPA .P. jirovecii revealed by this specific mutation in SOT recipients. It is noteworthy that SOT recipients T3 and T9 harboured allele IMPDH-3 (characterised by the G1020A mutation resulting in Ala261Thr) in combination with allele IMPDH-1, whereas the remaining eight SOT recipients harboured IMPDH-3 solely. These mixed infections may be explained by an in-progress process of selection of P. jirovecii organisms under MPA pressure, wild P. jirovecii strains being not entirely replaced by a predominant mutant P. jirovecii strain. While MPA was initially considered to protect against P. jirovecii in the 1990s [P. jirovecii populations, selection of specific strains characterised by potentially lower sensitivity to MPA and acquisition/transmission of these specific strains among susceptible SOT recipients.Consequently, MPA may have favoured the emergence of specific strains of he 1990s , an incrhe 1990s . At preshe 1990s . This paP. jirovecii selection under drug pressure has already been reported with the description of specific genotypes/phenotypes of dihydropteroate synthase (DHPS) in patients treated with sulfonamides that target the DHPS [P. jirovecii DHPS mutants [P. jirovecii DHPS mutants [P. jirovecii transmission from infected, treated patients to susceptible, untreated patients. Similarly, we recently described a P. jirovecii mutant of the gene of cytochrome b (CYB), which is the target of atovaquone [P. jirovecii circulation within human communities.The phenomenon of the DHPS ,29,30. P mutants ,29,30. T mutants ,29. Thisovaquone . This muovaquone . For theIMPDH-3 (characterised by the G1020A mutation resulting in Ala261Thr) and the low diversity of the P. jirovecii impdh gene in SOT recipients suggest a common identical source of the fungus in this patient group. Conversely, the high diversity of impdh gene, as well as the absence of the IMPDH-3 allele suggest several different sources of P. jirovecii in the control group. This diversity resulted in the identification of nine SNPs and eight alleles.The presence of allele impdh gene analysis in contributing to a method of P. jirovecii genotyping, we evaluated the Hunter index at 0.6952 (<0.95) [P. jirovecii isolates in a genotyping approach based on this sole locus. Nonetheless, impdh gene analysis could be added to the highly discriminative multilocus sequence typing (MLST) method combining mtLSUrRNA, CYB and superoxide dismutase gene analysis, that we have used to investigate Pneumocystis case clusters in hospital [Impdh gene analysis could provide an additional marker of P. jirovecii circulation among SOT recipients who are specifically at risk for PCP occurrence in a context of nosocomial acquisition of P. jirovecii and PCP outbreaks. MLST analysis including impdh gene may strengthen the concept of acquisition and transmission of specific P. jirovecii strains in SOT recipients, which has previously been discussed [Considering the potential of  (<0.95) . This vahospital . Impdh giscussed ,34.P. jirovecii strains under selection pressure that circulate in the SOT recipient population. A multicentre study, combining the enrolment of patients from diverse geographical regions and the genotyping of P. jirovecii specimens based on the aforementioned MLST approach is now warranted.To sum up, we pointed out a missense mutation Ala261Thr in SOT recipients treated with MPA, whereas this change was not found in a control group without prior exposure to MPA. This mutation may represent the signature of MPA exposure and the selection of specific


Purpose:Prunella vulgaris (PV), a traditional Chinese medicine, has been used to treat patients with thyroid disease for centuries in China. The purpose of the present study was to investigate its bioactive ingredients and mechanisms against Hashimoto’s thyroiditis (HT) using network pharmacology and molecular docking technology to provide some basis for experimental research.Methods: Ingredients of the PV formula were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Additionally, HT-related genes were retrieved from the UniProt and GeneCards databases. Cytoscape constructed networks for visualization. A protein–protein interaction (PPI) network analysis was constructed, and a PPI network was built using the Search Tool for the Retrieval of Interacting Genes (STRING) database. These key targets of PV were enriched and analyzed by molecular docking verification, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment.Results: The compound–target network included 11 compounds and 66 target genes. Key targets contained Jun proto-oncogene (JUN), hsp90aa1.1 (AKI), mitogen-activated protein kinase 1 (MAPK1), and tumor protein p53 (TP53). The main pathways included the AGE-RAGE signaling pathway, the TNF signaling pathway, the PI3K–Akt signaling pathway, and the mitogen-activated protein kinase signaling pathway. The molecular docking results revealed that the main compound identified in the Prunella vulgaris was luteolin, followed by kaempferol, which had a strong affinity for HT.Conclusion: Molecular docking studies indicated that luteolin and kaempferol were bioactive compounds of PV and might play an essential role in treating HT by regulating multiple signaling pathways. Prunella vulgaris (PV) is a perennial herbaceous plant in the genus Prunella1. It is a Chinese medicine widely used to treat inflammation, eye pain, and headaches   and thyroglobulin antibody (TG-Ab) (PV might exert its anti-inflammatory effects are poorly understood.eadaches . The antne (TCM) . Current (TG-Ab) . HoweverHashimoto’s thyroiditis (HT) is a genetic autoimmune disorder characterized by the destruction of thyroid cells by cell- and antibody-mediated immune responses . In deveAdditionally, the association between vitamin D deficiency, HT pathogenesis, and thyroid hypofunction has been demonstrated in several studies . TherefoNetwork pharmacology, combined with pharmacology and pharmacodynamics, is a novel research field that clarifies numerous compounds’ synergistic effects and underlying mechanisms by analyzing various networks of complex and multilevel interactions . The stuPV were obtained from the Traditional Chinese Medicine Systems Pharmacology Database, the Analysis Platform  database, and subsequent network pharmacology (The active constituents of macology . The TCMmacology . The nammacology . In thismacology .PV were retrieved from both TCMSP databases (http://lsp.nwsuaf.edu.cn/tcmsp.php), including 6,511 drug molecules and nearly 4,000 targets as well as the interaction between them (https://www.uniprot.org).The potential targets for the components of een them . The Unieen them . Gene inhttps://www.genecards.org/) and selection according to the criterion of RiskScore >1, the Therapeutic Target Database , OMIM (https://omim.org/), PharmGkb (https://www.pharmgkb.org/), and DrugBank (https://www.drugbank.ca/) were used to collect information on HT related to target genes. The association of PV with HT was then gathered as the core targets of PV for HT.The GeneCards database (http://www.cytoscape.org/)  . Moreovepe.org/) . In the pe.org/) . The toppe.org/) . The larPV for HT treatment were imported into the STRING database (https://string-db.org/) (The candidate targets of db.org/)  to constdb.org/) . Degree Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed in R using the ClusterProfiler package, and p. adjust (FDR) < 0.05 was considered statistically significant .AKI) (PDB ID: P31749) (http://www.rcsb.org/pdb). The protein structure was processed by AutoDock Tools (https://pubchem.ncbi.nlm.nih.gov/) as. sdf. Luteolin and kaempferol were vital active compounds, and AKI and MAPK1 were considered the major targets. Molecular docking of the ligands luteolin and kaempferol to the AKI and MAPK1 protein active sites was carried out using AutoDock Vina 1.1.2. Each docking calculation generated 20 structures, and the molecular docking output was prioritized based on the frequency of the possible ligand-binding site and a free energy score. Finally, the best possible conformations and visualized using Discovery Studio Visualizer 2.5 and PyMOL software .The three-dimensional (3D) molecular structures of hsp90aa1.1 ( P31749)  and mito P31749)  were dowck Tools  to removPV, and the Venn diagram of PV targets is shown in PV containing 11 compounds are shown in JUN), AKI, MAPK1, and tumor protein p53 (TP53) are shown in Of these, 923 were targets of the herbs comprising The herb–compound–target network contained 77 nodes (11 compounds and 66 genes), as shown in PV-related targets, and 17 intersection targets were identified . The relative enrichment analysis showed the following pathways: the AGE-RAGE signaling pathway in diabetic complications, the TNF signaling pathway, the PI3K–Akt signaling pathway, pathways in cancer, the MAPK signaling pathway, and the cancer-associated pathways, as shown in To better understand the function of intersecting genes, we conducted an enrichment analysis of Gene Ontology (GO). The top 10 biological processes mainly an inflammatory response, a cell-to-cell reaction, and a metabolic process are ranked in AKI and MAPK1 proteins have a stronger affinity for HT disease molecules. Luteolin (MOL000006) was considered as the uppermost active ingredient of PV against HT. Meanwhile, there was also a strong association between kaempferol (MOL000422) and AKI with MAPK1. The AKI and MAPK1 protein active pockets showed that the small molecules luteolin and kaempferol have a compact binding pattern  is usually manageable by levothyroxine (L-T4) administration, reducing the thyroid volume and supplementing the lack of hormones. Despite thyroid hormone replacement, some euthyroid patients with HT will continue to experience persistent symptoms that reduce their quality of life. uding HT . In addirophages . This stPV: luteolin, quercetin, beta-sitosterol, kaempferol, stigmasterol, morin, spinasterol, delphinidin, vulgaxanthin-I, poriferasterol monoglucoside_qt, and stigmast-7-enol. Molecular docking showed that the active ingredients, including luteolin and kaempferol, had a good affinity for the hub disease proteins in clinical therapeutics  signaling pathway, and some related to thyroid cancer. The present study identified JUN, AKI, MAPK1, and TP53 hub genes using the PPI network analysis. These results indicated that response . Luteoliresponse . A previresponse . Severalresponse . Luteoliresponse . The keyMeanwhile, the MAPK signaling pathway plays an important role in developing thyroid carcinoma, including cell proliferation and cell survival. The PI3K/AKT signaling pathway plays a key role in regulating the activation of inflammatory response cells and releasing inflammatory transmitters to the chronic inflammatory response in HT . By reguPV and HT is key towards therapeutic discovery and development.The present study included some limitations. First, some further verified experiments need to be conducted. Second, their specific molecular docking methods and locations need to be verified by further experiments. A comprehensive understanding of the key gene target of PV, which indicated that they might play an essential role in treating HT. PV may act against HT mainly through the TNF signaling pathway, the MAPK signaling pathway, and the PI3K–Akt signaling pathway.In summary, the network pharmacology and molecular docking show that luteolin and kaempferol were the main active components of