Sulfolobus acidocaldarius is a strictly aerobic crenarchaeon thriving in a thermoacidophilic environment, and its membrane is dominated by GDGTs. Here, we comprehensively analyzed the lipidome and transcriptome of S. acidocaldarius in the presence of terbinafine. Depletion of GDGTs and the accompanying accumulation of DGDs upon treatment with terbinafine were growth phase-dependent. Additionally, a major shift in the saturation of caldariellaquinones was observed, which resulted in the accumulation of unsaturated molecules. Transcriptomic data indicated that terbinafine has a multitude of effects, including significant differential expression of genes in the respiratory complex, motility, cell envelope, fatty acid metabolism, and GDGT cyclization. Combined, these findings suggest that the response of S. acidocaldarius to terbinafine inhibition involves respiratory stress and the differential expression of genes involved in isoprenoid biosynthesis and saturation.Cellular membranes are essential for compartmentalization, maintenance of permeability, and fluidity in all three domains of life. Archaea belong to the third domain of life and have a distinct phospholipid composition. Membrane lipids of archaea are ether-linked molecules, specifically bilayer-forming dialkyl glycerol diethers (DGDs) and monolayer-forming glycerol dialkyl glycerol tetraethers (GDGTs). The antifungal allylamine terbinafine has been proposed as an inhibitor of GDGT biosynthesis in archaea based on radiolabel incorporation studies. The exact target(s) and mechanism of action of terbinafine in archaea remain elusive.  Sulfolobales utilize the classic mevalonate pathway (MVA) for this step . GGP. GGPSulfaldarius  1]. The. TheSulfaldarius  11]. Th. ThSulfoS. acidocaldarius predominantly consists of monolayer-forming GDGTs with a lower abundance of GDNTs . ThiThermoplasma acidophilum have indicated the accumulation of DGDs upon treatment with terbinafine, a squalene epoxidase inhibitor in fungi −) was observed in exponential and stationary phase cells (m/z 715.507 [M-H]−) and archaetidic acid  were slightly elevated during the exponential phase; however, they were depleted during the stationary phase  .,27.S. acse cells . The levry phase  .m/z 1866.433–1854.339 [M-H]−), penta-hexose inositol phosphate GDGT-0 to 6 , di-hexose GDGT—0 to 6 , and GDGT- 0 to 6  was observed only in the stationary phase : aceA (isocitrate lyase), argG-H (argininosuccinate synthase and lyase), leu2 , and saci_2320 (predicted glutamate synthase)   28]. In. InS. acp-adjusted value < 0.05) were mapped to the arCOG pathway ‘Energy production and conversion’, as illustrated by the column graph  and NADH complexes responsible for reducing quinones . Mo. MoSulfobinafine . For CQHbinafine C [21]. Tbinafine C, which Archaeoglobus fulgidus was shown to alter the MK saturation profile in Escherichia coli [S. acidocaldarius is a homolog of this enzyme with 29% sequence identity. The transcript levels of this gene were unaffected. GGPP is a proposed common precursor of isoprenoids such as CQs and membrane lipids [S. acidocaldarius.An MK-specific reductase from hia coli . WP_0112e lipids . The tragrsB) transcript levels, which has been identified as a GDGT cyclization enzyme in S. acidocaldarius [grsB [egsA (saci_0640), which is responsible for the NAD(P)H- dependent reduction of dihydroxyacetonephosphate (DHAP) to produce a glycerol-1-phosphate backbone for archaeal lipids  [S. acidocaldarius harbors five such uncharacterized paralogs to GGR which are clustered into arCOG00570. Thus, these paralogs could be responsible for altering the saturation profile of the CQs.Terbinafine inhibits squalene epoxidase in fungi . Squalenolobales ,21,32,48nes (MK) . This clnes (MK) . S. acidRsa cluster has predicted acyl-CoA dehydrogenases, 3-hydroxyalkyl-CoA-dehydrogenase, and acetyl-CoA acetyltransferase in S. acidocaldarius which are involved in the synthesis of acetoacetyl-CoA, a precursor of the alternate mevalonate pathway in archaea [The Fad archaea ,49. The S. acidocaldarius, as previously observed for T. acidophilum [Sulfolobus, and its amounts are consistent across growth phases [T. acidophilum based on radiolabeling assays [Methanosarcina acetivorans shows a preference for the fully saturated diether species in a lipid extract [Our study confirms the growth phase-dependent depletion of GDGTs in the presence of terbinafine in dophilum . Signifih phases ,17. Unsag assays . Remarka extract .Rsa cluster, respiratory chain complex, grsB, archaellum, and slaA in the cell envelope indicate that terbinafine induces a multitude of responses in S. acidocaldarius related to energy metabolism. The synthesis of tetraether lipids and formation of cyclopentane rings are key adaptations that regulate rigidity and homeostasis [S. acidocaldarius. The presence of unsaturated CQs in the membrane, along with the upregulation of genes involved in the respiratory complex, indicates that the flow of electrons is disrupted. Furthermore, the altered expression of the F1F0-ATPase subunits suggests a possible problem with ATP synthesis in cells. A possible explanation for these membrane adaptations and transcriptome responses to terbinafine is that the organism adjusts to this respiratory stress by redirecting its metabolic energy to increase motility through the archaellum and not by investing its net reducing power in the saturation of CQs or synthesizing complex GDGTs. However, proteomics analysis and assessment of the impact of terbinafine on the respiratory capabilities of the organism may further support this conclusion.The archaeal membrane maintains metabolic processes through energy-transducing processes such as the transfer of electrons/protons or ATPase-dependent translocation of ions . The alteostasis ,51,52,53eostasis . SlaA iseostasis . An eleveostasis . This coS. acidocaldarius to terbinafine. Terbinafine has a multitude of effects on S. acidocaldarius, one of which is the altered DGD: GDGT ratio in the membrane. Considering its effects on the respiratory chain and saturation of CQs, terbinafine appears to be an indirect inhibitor of GDGT biosynthesis. The role of other isoprenoids in archaeal membrane architecture, and their regulatory mechanisms remain elusive. Furthermore, knowledge of quorum sensing in S. acidocaldarius is limited to lactonase, and the sensors responsible for membrane adaptations are not known [Sulfolobus membrane so far, making it difficult to speculate on any possible structural role of these isoprenoids. This is the first study to show that an allylamine drug—like terbinafine—affects the saturation profile of respiratory quinone (CQ) in the membrane of S. acidocaldarius. This drug causes respiratory stress in organisms, causing a redirection of its metabolism towards energy conservation and motility. This could explain the reduced levels of complex phospholipids, such as GDGTs, in the membrane. This study describes the cellular response of ot known . TherefoS. acidocaldarius MW001 was grown at 75 °C by shaking at 120 rpm in Brock medium, pH 3 supplemented with 0.1% NZ-amine, 0.2% dextrin, and 10 µg/mL uracil. As indicated, various amounts of a stock solution containing 250 mg/mL terbinafine hydrochloride  were grown in duplicate until the exponential phase (OD600 nm of 0.3) and then subjected to terbinafine (0.31 mM and 1.22 mM) or DMSO treatment (0.16%) for 3 h. Cultures were harvested , and cells were processed further to isolate RNA using the TRIzol method [S. acidocaldarius DSM639 genome (RefSeq ID: NC_007181.1) using STAR [p-values (<0.05) were used for data analysis to reduce the false discovery rate (FDR). The results of differential gene expression from DESeq2 were mapped to the archaeal cluster of orthologous genes (arCOGs) and then manually revised [For RNA isolation, l method . The obting STAR . These ring STAR . Differeing STAR . Adjuste revised . FiguresS. acidocaldarius MW001 cultures were inoculated in 200 mL of Brock medium supplemented with either 0.16% DMSO or 1.22 mM terbinafine in biological triplicates. Cultures were harvested by centrifugation  at the exponential (22 h) and early stationary phases (40 h) of growth. The pellets were washed with fresh Brock medium, centrifuged again, flash-frozen in liquid nitrogen, and freeze-dried at 0.07 mbar, −55 °C for 48 h. Freeze-dried biomass (10 mg) was processed for lipid extraction using a modified acidic Bligh and Dyer method [2 stream to form a lipid film, re-extracted with chloroform, and dried again [Sulfolobus lipids were separated using an Acquity UPLC CSH C18 column  with an eluent flow rate of 300 µL/min at 55°C. A scan range of m/z 125–2500 was used in the full MS mode. The voltage parameters were 3 kV (spray), −75 V (capillary), −190 V (tube lens), and −46 V (Skimmer). Eluent A was MilliQ water : MeCN (40:60), and Eluent B was MilliQ water: MeCN:1-BuOH (0.5:10:90), both containing 5 mM ammonium formate (pH 6.5) [r method  using 0.ed again . This sted again . The obt(pH 6.5) ,63. Stat(pH 6.5) .


Staphylococcaceae bacteria composition of the nasal flora in dromedary camels and evaluate the presence of methicillin-resistant Mammaliicoccus (MRM) and methicillin-resistant Staphylococcus (MRS) in dromedary camels in Algeria. Nasal swabs were collected from 46 camels from seven farms located in two different regions of Algeria (M’sila and Ouargla). We used non-selective media to determine the nasal flora, and antibiotic-supplemented media to isolate MRS and MRM. The staphylococcal isolates were identified using an Autoflex Biotyper Mass Spectrometer (MALDI-TOF MS). The mecA and mecC genes were detected by PCR. Methicillin-resistant strains were further analysed by long-read whole genome sequencing (WGS). Thirteen known Staphylococcus and Mammaliicoccus species were identified in the nasal flora, of which half (49.2%) were coagulase-positive staphylococci. The results showed that four out of seven farms were positive for MRS and/or MRM, with a total of 16 isolates from 13 dromedary camels. The predominant species were M. lentus, S. epidermidis, and S. aureus. Three methicillin-resistant S. aureus (MRSA) were found to be ST6 and spa type t304. Among methicillin-resistant S. epidermidis (MRSE), ST61 was the predominant ST identified. Phylogenetic analysis showed clonal relatedness among M. lentus strains, while S. epidermidis strains were not closely related. Resistance genes were detected, including mecA, mecC, ermB, tet(K), and blaZ. An SCCmec type VIII element was found in a methicillin-resistant S. hominis (MRSH) belonging to the ST1 strain. An SCCmec-mecC hybrid element was detected in M. lentus, similar to what was previously detected in M. sciuri. This study highlights that dromedary camels may be a reservoir for MRS and MRM, and that they contain a specific set of SCCmec elements. This emphasizes the need for further research in this ecological niche from a One Health perspective.Dromedary camels are an important source of food and income in many countries. However, it has been largely overlooked that they can also transmit antibiotic-resistant bacteria. The aim of this study was to identify the  Camelus dromedarius) are selectively bred, raised, and grazed for their milk and meat [Dromedaries  and MRM [S. aureus [Mammaliicoccus and Staphylococcus is mediated by the production of either an alternative penicillin-binding protein with low affinity for β-lactams or a β-lactamase [blaZ gene, hydrolyses only penicillin-rings, hence conferring narrow-spectrum β-lactam resistance [mec gene [mec genes are known . The mecA and mecC genes can be acquired by horizontal gene transfer through staphylococcal cassette chromosome mec (SCCmec) elements in Staphylococcus and Mammaliicoccus. The mecB and mecD genes have been found in Macrococcus on SCCmec elements, as well as on unrelated mecD-carrying Macrococcus resistance islands (McRImecD) and large mecB-carrying plasmids [mecB-positive staphylococci (S. aureus) has been reported [mec types have been described in MRSA [mec structures in Staphylococcus became more apparent with the extensive use of WGS and non-aureus staphylococci and mammaliicocci have been suggested as possible reservoirs of SCCmec elements [mec element is a 20–70 kb genetic element inserted at a specific site  in orfX  near the replication origin of the chromosome [mecC has been identified as a second determinant for methicillin resistance in Staphylococcus species. It was described in MRSA from humans [mecA of S. aureus N315, this variant was designated as mecC and is encoded in MRSA within a distinct SCCmec classified as type XI [Little is known about the clinical importance of methicillin-resistant non- and MRM ,20. Howe. aureus ,21. Resiactamase . The β-lsistance . The altmec gene . To dateplasmids , and onlreported . Current in MRSA . The diselements . The SCCromosome ,29. Sincm humans ,31 and dm humans  in Denmam humans  and onlym humans . Sharing type XI .mecC in staphylococci from animals and food [Staphylococcaceae in camels and their antimicrobial resistance and virulence [In Africa, though less well known, antimicrobial resistance (AMR) is a growing concern in humans and animals . The sitand food . Despiteand food . The nasand food . To our irulence . In AlgeStaphylococcus and Mammaliicoccus species were identified. The results showed that coagulase-positive staphylococci (CPS) had the highest prevalence rate, accounting for 49.2% of the identified strains, followed by coagulase negative staphylococci (CNS) at 25.8%, while mammaliicocci accounted for 25.0% of the total strains.In this study, one hundred and twenty strains were isolated from forty-six healthy dromedaries in seven different farms in Algeria . ThirteeS. aureus was the most commonly detected, with isolation rates of 54.2%, followed by S. delphini at 30.5% and S. intermedius at 15.3%. S. aureus and S. delphini were found in all seven farms. M. sciuri was the only Mammaliicoccus species detected, with a prevalence of 25.0%, and it was detected on six farms, with the highest occurrence on farm 1 (n = 10), followed by farms 2 and 3 , while the remaining three were in the south of Algeria . On both farm 1 and farm 5, six out of ten dromedaries tested positive for MRS and/or MRM, resulting in a proportion of positive animals on these farms of 60%.M. lentus (n = 6), S. epidermidis (n = 6) S. aureus (n = 3), and S. hominis (n = 1).Sixteen MRS and MRM were recovered from thirteen different dromedaries distributed over the four positive farms. The identified species were spa type t304. Among methicillin-resistant S. epidermidis (MRSE), ST61 (n = 3) was the predominant ST. Other MRSE STs were ST54, ST640, and ST35, represented by one strain each. The MRSH was ST1. For the other species, no MLST scheme is available.First, all assembled genomes from MRS/MRM isolates (n = 16) were quality checked using CheckM and pubMLST to assess genome completeness and perform species identification, respectively. The results of the MALDI-TOF MS classification and WGS-based classification using ribosomal genes  was in concordance with each other. An overview of the species identification of both methods can be found in S. epidermidis strains exhibited a greater degree of variation in this study and differed by 506 to 31,322 SNPs. Within ST61, the SNP difference was 506 to 546. As for M. lentus, five strains displayed clonal relatedness with a small range of SNP differences, from 9 to 47 SNPs, while one strain differed significantly, with over 4000 SNPs.Analysis of the relatedness of the three isolated MRSA ST6/t304 using single nucleotide polymorphisms (SNPs) revealed that the strains were highly similar, with only two to five SNPs differentiating them. The mecA, and in 5 M. lentus mecC was detected in addition to mecA. The other resistance genes present were responsible for resistance against penicillins (blaZ), aminoglycosides (aadD), trimethoprim (dfrG), lincosamides and macrolides (erm(B), erm(A), msr(A), mph(C)), fosfomycin (fosB), fuscidic acid , and tetracycline (tet(K)). The MRSA strains carried only genes responsible for resistance to β-lactams , whereas MRNaS also carried other resistance genes. The bleO gene, responsible for bleomycin resistance, was detected only in MRSH.Thirteen different antimicrobial resistance genes were identified by WGS in the 16 examined isolates . All strS. epidermidis strains isolated from camels, the ten available strain sequences were examined ) was detected in eight isolates, with six strains from our study and two from Akarsu et al. [mecA was found in six strains from our study and one strain from the other study. The gene fusc, which mediates resistance to fuscidic acid, was detected in the isolates from the study of Akarsu et al. [fusB was present in four (67%) strains from our study. Moreover, the dfrG gene was detected in only one strain among those isolated in this study (SAMN33407038).To compare the distribution of AMR genes in examined . This inu et al. . Additiou et al. , whereashlgA, hlgB, and hlgC, which encode the γ-hemolysin; sak, which encodes a staphylokinase; scn, which is associated with immune evasion; aur, which encodes aerolysin, the sea gene encoding enterotoxin; and the lukC and lukD genes, which encode a leukotoxin. The icaABC and its regulator icaR, which are responsible for biofilm formation, mucus production and its regulation, and facilitating attachment to environmental surfaces, were found in all MRSA strains and in one MRSE isolate (SAMN33407038).A total of sixty-four virulence genes were found among the three MRSA ST6/t304 strains, while in MRSE twenty-eight virulence genes were identified. These genes encode factors for adherence, exoenzymes, immune evasions, exotoxins, effector delivery, and stress survival . In the S. epidermidis strains showed that the ten strains carried a total of forty-three virulence genes  [mecA was typed as SCCmec type VII and contains mec complex , a cad operon (cadADC), encoding cadmium resistance and ccrA1/ccrB5 recombinases. The mecC region was part of a homologous class of E mec gene complex (mecI-mecR1-mecC-blaZ) (Genome sequencing of these five G515014) . The SCCcC-blaZ) .Staphylococcus and Mammaliicoccus. By non-selective isolation, we found 13 distinct species of Staphylococcaceae. Most of the strains were CPS (49.2%) with identification of S. aureus, S. delphini, and S. intermedius. The CPS group is commonly associated with severe infections [S. aureus in nasal carriage, ranging from 6.2% to 89.1% [This is the first study specifically investigating MRS and MRM in dromedary camels. Because little is known on the nasal staphylococcal flora, we also included non-selective isolation of fections . Earlierto 89.1% ,41. The S. aureus is a common nasal colonizer of healthy camels with, however, significant variation in prevalence. The frequency of S. aureus isolated from camels in our study is similar to other studies conducted in Nigeria and higher than a recent study conducted in Tunisia [S. agnetis, S. arlettae, S. ureilyticus, S. delphini, S. felis, M. sciuri, M. lentus, S. muscae-like, S. pasteuri, S. chromogenes, S. schleiferi, S. epidermidis, S. hominis, and S. simulans [S. gallinarum, S. lutrae, S. intermedius, S. hyicus, and S. xylosus in dromedary camels. Further studies are necessary to understand the staphylococcal flora of the nose of camels.Overall, these findings suggest that  Tunisia ,43. Prevsimulans ,42. To oM. lentus and S. epidermidis, which confirms the findings of Silva et al. (2022) [M. lentus as the most prevalent methicillin-resistant species. These species are the most prevalent coagulase negative staphylococci at the clinical level and as part of the normal nasal microbiota of healthy individuals, which may suggest a possible human origin [M. lentus were found in either study. Further research is needed to fully understand the prevalence and mechanisms of methicillin resistance in M. lentus. Our results on the prevalence of MRS will also allow us to set up a surveillance with a more accurate sample size estimation.In the past decades, there has been a growing concern regarding the issue of the staphylococci of animals, especially related to methicillin resistance. Thorough investigations into MRS/MRM in camels are infrequent, particularly those that involve nasal carriage, nor have there been a lot of strains subjected to WGS. Our results indicate that more than half of the farms tested were positive for MRS/MRM, though there seem to be regional differences. We found a high diversity of species and clones, indicating that methicillin resistance is widespread. Similar findings has been reported from Kenya and Saudi Arabia ,44. The . (2022) , which an origin ,46. No smecA carried on SCCmec elements is considered to originate from members of the Mammaliicoccus group , and the members of this group played a major role in the evolution and dissemination of the mecA gene [M. lentus strains carried mecA and mecC genes; these strains were very similar and support a clonal spread of this resistant bacterium. The mecC was found encoded within a hybrid SCCmec element comprised of a mecA encoding SCCmec type VII and an mecC region. This element has been described in three M. sciuri, originally in GVGS2 from a caesarean incision wound in a Belgian Blue cow from England [mecC-carrying isolates were M. sciuri subsp. carnaticus, supporting the existence of a potential reservoir for mecC-carrying in M. sciuri [mec-mecC element has been reported in a single S. aureus isolate from a racehorse wound [mecA-mecC hybrid element was highly conserved, with the archetypal version reported originally in GVGS2 [The ecA gene . In this England , LP600 f England , and in  England . The mec. sciuri ,48,49. Ise wound . In eachin GVGS2 .mec has been identified in M. lentus, in dromedary camels, and in Algeria, while the molecular epidemiology of M. lentus in the farm environment was largely unknown. The origin of this element is yet to be uncovered. The mecC gene has been associated with wildlife, especially hedgehogs [mecC.It is the first time this hybrid SCCedgehogs ,52, and edgehogs . Most comec Iva, which is commonly found in this ST [Three MRSA strains identified as ST6/t304 were near identical, and all harboured an SCC this ST . This ST this ST ,55. The  this ST  and in p this ST  and rece this ST . This ra this ST . In Alge this ST ,60. The mec IIa, which was the most predominant SCCmec element in these strains. MRSE ST61 has been found in association with infections in humans [S. hominis subsp. hominis. The strain carried the SCCmec VIII.In MRSE, ST61 was the most predominant ST identified in dromedaries with SCCblaZ and mec genes. It is worth noting that previous studies on MRSA ST6/t304 strains had identified various other genes, including ermC, tet(K), fusC, lnuA, and aadA [M. lentus) harboured the blaZ gene. These results are consistent with a previous study in Kenya where all the isolates from dromedary camels harboured the blaZ gene [tet(K) gene was highly prevalent. This gene is commonly found in Staphylococcus and Mammaliicoccus species, including S. epidermidis [tet(K) gene can be a concern in African countries, where tetracycline antibiotics are commonly used for treatment due to their affordability and availability.All MRNaS isolates analysed in this study had additional resistance genes, in contrast to the MRSA ST6/t304 strain that only had the and aadA . Most stlaZ gene . Apart fdermidis . The prebleO) was detected in MRSH ST1 isolates. The bleomycin is commonly used for the treatment of various cancers, but it is not a commonly used antibiotic for bacterial infections [bleO gene is related to selective pressure from the use of bleomycin in the treatment of bacterial infections. Instead, it is more likely that the bleO gene was acquired through horizontal gene transfer from other bacteria in the environment that have been exposed to bleomycin [Interestingly, the bleomycin resistance gene . In both studies, the camels were in traditional farming communities, and the antibiotics may not be widely available or affordable, leading to limited or no use of antibiotics in treating bacterial infections, and thus a low selective pressure is assumed. However, there are few studies available on the general level of antimicrobial use in dromedaries or antimicrobial resistance in bacteria from dromedaries, and the interpretation remains difficult [Comparison of distribution of antimicrobial resistance genes among camel-derived u et al.  showed tifficult ,41.S. epidermidis showed that the strains harboured different virulence genes that were not similar. The fact that the S. epidermidis strains isolated from both camel studies have different characteristics suggests that these strains may have adapted to the host and developed a distinct set of virulence factors.Different virulence-encoding genes have been detected in MRSA and MRSE strains with little variation. These virulence factors are associated with several key functions, including adherence, exoenzyme production, exotoxin production, effector delivery, and stress survival, all of which are important for pathogenicity. The result that MRSA ST6/t304 isolates were negative for Panton–Valentine leukocidin (PVL) is consistent with previous findings in northern Europe and Asia ,55. Simimec-mecC, suggest that dromedaries may serve as a reservoir for antibiotic-resistant staphylococci, potentially leading to zoonotic transmission to humans. As such, this research highlights the importance of continued surveillance and monitoring of antibiotic resistance in both animal and human populations to mitigate the spread of antibiotic-resistant bacteria and preserve the effectiveness of antimicrobial therapies.The finding that dromedary camels in Algeria harbour MRS and MRM, as well as the hybrid element SCCCamelus dromedarius) were sampled from seven Algerian farms present in two different regions (three farms located in the centre of Algeria (M’sila) and four farms in the south (Ouargla)) ) . We colluargla)) . These dStaphylococcaceae were first enriched in 6.5% NaCl Brain Heart Infusion (BHI) broth  at 37 °C overnight, and then sub-cultured onto Columbia agar  supplemented with 5% sheep blood, after which the plates were incubated at 37 °C for 24–48 h. Colonies that resembled staphylococci or mammaliicocci were chosen based on their distinct morphologies, and purified on Columbia agar with 5% sheep blood. The isolates were grown at 37 °C for 24–48 h, and subsequently stored at −80 °C in 50% BHIB and 50% glycerol until further analysis.Staphylococcaceae involved some adjustments to the previously described protocol [The method used for isolating methicillin-resistant protocol . FirstlyStaphylococcus and Mammaliicoccus using the catalase test. Subsequent species identification was performed using the Autoflex Biotyper Mass Spectrometer (MALDI-TOF MS)  via the direct transfer method with α-cyano-4-hydroxycinnamic acid as the matrix, following the manufacturer’s protocol. If no peaks were detected, the samples were retested using formic acid treatment. The resulting spectra were analysed using the MBT (MALDI BioTyper) compass software version 4.1.  as previously described [The isolated bacteria were presumptively identified as escribed .mecA gene was performed as described previously [mecA were subsequently tested for the presence of the mecC gene. The primers used are listed in One strain per species per animal was selected for PCR. The DNA was extracted as previously described  using lyeviously . Strains−1; Promega, Madison, WI, USA). Bead bashing was performed with a TissueLyzer  twice for 5 min at 30 oscillations per minute. The DNA quality was verified using a Nanodrop Spectrophotometer. If A260/A230 or A260/A280 were below 1.7, DNA was further cleaned using CleanNGS  magnetic beads in a 1:1 ratio. High-quality HMW DNA was subjected to a rapid library preparation  with 400ng DNA input per isolate. A maximum of 12 isolates were multiplexed on a single R.9.4.1 flow cell. Sequencing was carried out on a GridION device (ONT), allowing raw database calling and demutiplexing  using the MinKNOW software (ONT). An overview of sequencing output and coverage can be found in Sixteen mec-positive isolates were selected for WGS using Oxford Nanopore Sequencing (ONT), as previously described ,70. Colohttps://github.com/rrwick/Filtlong, accessed on 10 October 2022), prior to subsampling into 10 subsamples (--min_read_depth 50 --count 10 --genome_size 5M) [https://github.com/rrwick/Minipolish, accessed on 10 October 2022), or wtdbg2 v1.12 [Default settings were used to generate consensus genomes in Trycycler, including removal of contigs with coverage below 10% of the median sample sequencing coverage, clustering, reconciling, multiple sequence alignment, read partitioning, and generation of complete circular genome and plasmid consensuses. Final outputs were still subjected to read mapping and polishing using minimap2 v2.20 [Staphylococcus genomes. When all marker genes were identified, a completeness of 100% was reported. The assembled contigs were annotated using the automated RAST server (Rapid Annotation using Subsystem Technology) [The fastq files were used in an in-house bacterial whole genome assembly pipeline using the Trycycler pipeline v0.5.3 . In shorsize 5M) . Each susize 5M) , raven vsize 5M) , miniasmg2 v1.12 , as instp2 v2.20  and medap2 v2.20  and Checp2 v2.20 . A genomhnology) .spa typing was carried out with SpaTyper v1.0, both of which are available through the Centre for Genomic Epidemiology . The CSI phylogeny 1.4 tool available on the Centre for Genomic Epidemiology (CGE)  was used, with default parameters for phylogenetic analysis. The resulting phylogenetic trees were visualized using iTOL v6.3.2. [Multilocus sequence typing (MLST) was conducted using the MLST Finder v2.0.4, and  v6.3.2. .https://cge.cbs.dtu.dk/services/ResFinder/, accessed on 12 October 2022). Virulence factors were detected using VirulenceFinder 2.0  and the virulence factor database (VFDB) [mec typing scheme was used to assign MRS isolates to the appropriate subtype using the SCCmecFinder v1.2 online tool, available through the Centre for Genomic Epidemiology  and blast for the hybrid SCCmec and visualized using EasyFig.The assembled files were analysed for the presence of resistance genes using ResFinder v.3.0 (e (VFDB) . The SCCStaphylococcaceae nasal flora in dromedary was further characterized, and five species that were never reported before in dromedaries were found. Nearly half of the species detected were CPS, with S. aureus as the most frequent species. The prevalence of MRS/MRM at farm level was 57%, and M. lentus and S. epidermidis as the most frequent species. CA-MRSA was also detected, and the origin of these strains is probably human, indicating cross contamination between humans and dromedary camels. Similarly, most of the MRSE were ST61, typically found in human infections. The novel variant of the staphylococcal SCCmec type VIII element has been identified in MRSH. All but one of the M. lentus strains had the rare SCCmec-mecC hybrid element, indicating a specific epidemiology of this element in Algerian camel Staphylococcaceae. Moreover, this is the first time that this element was detected in M. lentus. These results are an indication for a specific epidemiology in Algeria, where mecC seems to be more associated with methicillin resistance than elsewhere in the world, though the low number of publications from that region needs further investigation. One Health-inspired research, including all compartments, on the prevalence and types of MRS and MRM are necessary to elucidate the epidemiology and spread of methicillin resistance, as this resistance compromises health by a large degree.In conclusion, the


Helicobacter pylori and Chlamydia. From secondary infections in cancer patients, microorganisms such as Pseudomonas aeruginosa, Escherichia coli, and Klebsiella species are commonly isolated. In general, these are highly diverse bacterial species and they have an exceptional ability to adapt and develop resistance against antimicrobial agents. Using an improved understanding of the pathogenic mechanisms of bacteria, it is necessary to review the topic of antimicrobial stewardship to optimize patient outcomes. Analysis of prophylactic antibiotic usage must also be addressed. Recent findings certainly support further examination of bacterial products that can be evaluated for their utilization as anti-cancer therapeutic agents.Bacterial involvement in cancer can be grouped into three categories: (i) direct association of bacteria in the processes of cancer development, (ii) secondary infection as a consequence of a patient’s weakened immune system, and (iii) utilization of bacteria in cancer therapeutics. Regarding a bacterial etiological or cancer-causing role, effective prevention strategies could be formulated after obtaining a precise understanding of the pathological mechanisms. Therefore, looking into the virulence factors of relevant bacterial species is critical in support of altering treatment plans with a proactive approach. This review has attempted to analyze both clinical/epidemiological and laboratory studies to understand bacterial mechanisms in cancer formation, particularly the virulence factors of Helicobacter pylori can directly impact gastric carcinogenesis. The Helicobacter pylori-associated virulence factor cytotoxin-associated gene A perhaps plays an important role through different mechanisms such as aberrant DNA methylation, activation of nuclear factor kappa B, and modulation of the Wnt/β-catenin signaling pathway. Many other bacteria, including Salmonella and Pseudomonas, can also affect Wnt/β-catenin signaling. Although Helicobacter pylori is involved in both gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma, its role in the latter disease is more complicated. Among other bacterial species, Chlamydia is linked with a diverse range of diseases including cancers of different sites. The cellular organizations of Chlamydia are highly complex. Interestingly, Escherichia coli is believed to be associated with colon cancer development. Microorganisms such as Escherichia coli and Pseudomonas aeruginosa are frequently isolated from secondary infections in cancer patients. In these patients, the common sites of infection are the respiratory, gastrointestinal, and urinary tracts. There is an alarming rise in infections with multidrug-resistant bacteria and the scarcity of suitable antimicrobial agents adversely influences prognosis. Therefore, effective implementation of antimicrobial stewardship strategies is important in cancer patients.In cancer development and its clinical course, bacteria can be involved in etiology and secondary infection. Regarding etiology, various epidemiological studies have revealed that  Pathogenic bacteria possess a large number of molecules and diverse mechanisms, which act as virulence factors and avert the host defense systems in order to initiate the disease processes. Apart from cell surface structures and metabolic arrangements, bacteria secrete several substances such as enzymes, toxins, and exopolysaccharides—many of these secreted biomolecules can enter the host cells and are able to control or damage the intracellular signaling/structures . All of Escherichia coli (E. coli), a Gram-negative, non-spore-forming, flagellated bacillus belonging to the family Enterobacteriaceae. Pathogenic E. coli strains are subdivided into different categories according to their clinical manifestations such as enteropathogenic (EPEC), necrotoxigenic (NTEC), and uropathogenic E. coli (UPEC). The prevalence of urinary tract infections (UTIs) is common in society and hospital settings, and UPEC is the leading pathogen in both uncomplicated and complicated UTIs [One of the most common infectious agents and also one of the most studied organisms is ted UTIs . Besidested UTIs . These vE. coli in inflammatory bowel disease could predispose colon cancer development [Enterobacteriaceae family—E. coli and Klebsiella spp., and another Gram-negative bacillus—Pseudomonas aeruginosa have been commonly isolated from cancer patients [Helicobacter pylori in gastric cancer, Salmonella typhi in gallbladder cancer, and Chlamydia pneumoniae in lung cancer [Enterobacteriaceae) and cytotoxin-associated gene A  can induce neoplasia by a number of mechanisms such as modulation of Wnt signaling pathway or maintaining an inflammatory state [Enterobacteriaceae family) are believed to promote colon cancer, but on the other hand these bacteria may be used as anti-cancer agents. The Salmonella genome encodes a wide range of virulence factors, which can alter various intracellular signaling pathways including the Wnt/β-catenin and transforming growth factor-β (TGF-β), thus contributing to tumorigenesis [P. aeruginosa such as mannose-sensitive hemagglutinin (PA-MSHA), cyclodipeptides, and exotoxin are being evaluated to be used in anti-cancer management [An intriguing aspect of certain bacterial virulence factors is their link with carcinogenesis. It is thought that chronic inflammation induced by elopment . Of notepatients ,6,7. In g cancer ,9. Bactery state . Interesigenesis . In contigenesis . Similarnagement ,13,14. AH. pylori  is a Gram-negative, flagellated, motile, and spiral-shaped/helical bacillus that grows in the gastric mucosa layer of more than 50% of the human population [H. pylori in 1982 by Barry J. Marshall and John R. Warren [H. pylori seropositive subjects [pulation . This ba. Warren , it took. Warren . Subsequsubjects ,19.H. pylori chronic infection [H. pylori can be divided into two strains, cagA-positive and cagA-negative; the former has a strong association with gastric cancer development. Furthermore, H. pylori’s direct action on gastric epithelial cells can cause the activation of nuclear factor kappa B (NF-κB), induction of mutations of tumor suppressor p53, and aberrant DNA methylation.In an interesting review, Chiba et al. described two major pathways for the development of gastric adenocarcinoma due to nfection . One of H. pylori infection, which again triggered the activation of another pro-inflammatory transcription factor—signal transducer and activator of transcription 3 (STAT3) [H. pylori-infected gastritis cases with 15 H. pylori-negative controls observed a distinctly enhanced NF-κB expression in the H. pylori-infected cases [H. pylori infection [H. pylori disrupted the p53 tumor suppressor pathway, and p53 alterations can be observed in lesions ranging from gastritis to gastric cancer [H. pylori-related lesions expressed the mutant-type p53 [H. pylori-infected cases [H. pylori-positive cases had p53 mutation [H. pylori infection resulted in p53 inhibition [H. pylori-linked chronic gastritis, gastric cancer, and cases with pre-neoplastic lesions followed up for 10 years, Compare et al. concluded that global DNA hypomethylation is an initial feature in H. pylori-linked gastric tumorigenesis [H. pylori-induced gastric cancer development [A key pro-inflammatory NF-κB pathway has been shown to promote the neoplastic processes of gastric mucosa. A study on AGS, SNU-1, and HGC-27 gastric cancer cell lines revealed that the activation of NF-κB occurred in  (STAT3) . Similar (STAT3) . Anothered cases . A numbenfection ,25,26. Ftype p53 . In addied cases . In the mutation . Studieshibition . On the hibition . After aigenesis . Intereselopment . Their sH. pylori-associated gastric cancer has a strong connection with gastritis, especially chronic atrophic gastritis of the corpus or type AB gastritis, where Th1-type CD4 cells and their product interferon-gamma (IFN-γ) may play a critical role, along with other pro-inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNFα). Apart from cagA, the bacterium also has a number of virulence factors, including vacuolating cytotoxin A (vacA), blood group antigen-binding adhesin (babA), and sialic acid-binding adhesin (sabA) [H. pylori infection [It is known that n (sabA) ,36. Thesnfection . Overallnfection .H. pylori-infected people, approximately 2–3% and 0.1% of individuals develop gastric adenocarcinoma and gastric MALT lymphoma, respectively [H. pylori strains are perhaps less virulent than those involved in gastric carcinoma [H. pylori infection could result in continued immune cell activation that might favor malignant transformation [H. pylori eradication therapy at the initial stage and for low-grade lymphoma is associated with satisfactory outcomes  and the fusion product is a powerful NF-κB activator [H. pylori eradication therapy should undergo alternative regimens such as chemotherapy and radiotherapy [Overall, the gastrointestinal tract is a common extranodal site for the development of lymphomas, although the incidence rate of primary lymphomas in the gastrointestinal tract is low and the majority of these lymphomas originate in the stomach . It has lignancy . In genectivator . In addictivator . Furtherotherapy . Regardiotherapy ,52.Histological features of gastric MALT lymphomas include lymphoepithelial lesions and the presence of centrocyte-like cells, monocytoid cells, immunoblast-like cells, and plasma cells ,54. MoreE. coli, Fusobacterium nucleatum, Bacteroides fragilis, and Streptococcus gallolyticus (Streptococcus bovis)   [61,62,6Chlamydia is unique. Chlamydiae are non-motile, Gram-negative, obligate intracellular bacteria, and exist in two morphologically distinct forms: elementary body (EB) and reticulate body (RB). The chlamydial cell wall consists of an outer membrane  and an inner cytoplasmic membrane [Chlamydia trachomatis infection, the target areas are usually the single-layer columnar cells or their transformation zone with the non-keratinizing squamous epithelium, which is typically found in the genital tract [The life cycle of membrane . The EB al tract . The EB al tract . On the Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci (zoonotic transmission). The latter two species can cause pneumonia. However, C. trachomatis is divided into 3 variant strains (biovars), which are again subdivided into several serotypes (serovars). Serovars A–C are responsible for trachoma (trachoma biovar), whereas the sexually transmitted serovars D–K  can cause genital tract infections and serious complications such as pelvic inflammatory disease, infertility, and ectopic pregnancy [C. trachomatis promotes human immunodeficiency virus infection and cervical cancer pathogenesis. On the other hand, the serovars L1–L3 (LGV biovar) causes lymphogranuloma venereum [Chlamydia infections have chronically been the most frequent notifiable sexually transmitted disease in the United States, with over 1.5 million cases reported in 2020 alone. More than 60% of all reported cases were among persons aged 15–24 and with most hosts being asymptomatic. Thus, understanding the long-term effects of this bacterium in terms of virulence, and the pathological consequences such as cancer development, is imperative.Three pathogenic species for humans are regnancy . In addivenereum . AccordiChlamydiae acquire the necessary nutrients. The bacteria are unable to produce the essential components of energy transduction and nucleic acid biosynthesis, as well as a number of amino acid biosynthesis pathways. So, they acquire these vital nutrients (including ATP) by selectively redirecting transport vesicles and capturing intracellular organelles [Chlamydia precisely, it is necessary to identify the relevant chlamydial virulence factors and their specific roles in disease severity.A vital step in chlamydial pathogenesis involves the mechanisms by which ganelles . Regardiganelles . These fC. trachomatis species [C. trachomatis serovar D strains in C3H/HeJ mice showed a reduced infectious capability compared to wild-type bacteria. Interestingly, Borges et al. revealed that CT135 impacted the expression of many proteins which are supposed to play a key role in bacterial virulence, including CT456/Tarp [Chlamydia spp. can manipulate the host cytoskeleton component actin to facilitate their invasion, replication, and disease spread. By restructuring the host actin cytoskeleton, the chlamydial type III-secreted ‘translocated actin recruiting phosphoprotein’ (Tarp) effector possibly supports bacterial entry into host cells [One of the most studied chlamydial virulence factors is plasmid glycoprotein 3 (Pgp3). Generally, a number of chlamydial species and strains carry a 7.5 kb plasmid that encodes 8 Pgps. However, it is assumed that the abovementioned plasmid protein is a contributing factor at least for the pathogenesis of  species . Moreove species . Their e456/Tarp . Of note456/Tarp . Chlamydst cells ,95.C. pneumoniae utilizes Pmp6, Pmp20, and Pmp21 [C. psittaci infection [C. pneumoniae Pmp 20 and Pmp 21 increased pro-inflammatory IL-6 and monocyte chemoattractant protein-1 via NF-κB pathway [C. pneumoniae Pmp21 can bind to EGFR and induce EGFR activation [C. pneumoniae infection has been shown to be associated with activation of different signaling molecules such as PI3K, mitogen-activated protein kinase, or ERK , LPS (a component of the outer membrane), secretion systems (which render toxins and enzymes into the host’s intracellular or extracellular space), secondary metabolites (such as phenazines like pyocyanin—responsible for greenish color), quorum sensing , and biofilm formation [ormation ,141. PatP. aeruginosa infections has become a serious problem because of the bacteria’s very high capability to develop resistance against several antibiotics. Furthermore, indiscriminate use of antibiotics favors the emergence of multidrug-resistant P. aeruginosa strains. In general, the principal mechanisms by which P. aeruginosa can resist antibiotic molecules may be categorized into three groups, i.e., intrinsic, acquired, and adaptive resistance [P. aeruginosa can survive and cause recurrent infections.The management of sistance . ExampleP. aeruginosa in cancer therapy. Since the initial observation of a bacterial anti-neoplastic role by German physician W. Busch in 1868 [P. aeruginosa-released anti-cancer substances such as exotoxin A, mono-rhamnolipids, and azurin [P. aeruginosa-linked anti-tumor mechanisms include the growth of bacteria in hypoxic regions of tumors, the production of toxins, modulation of host immune responses, and delivery of therapeutic genes that encode cytotoxic peptides or prodrug-converting enzymes [P. aeruginosa toxins are favorable biomolecules for the construction of recombinant immunotoxins or chimeric proteins  for anti-cancer therapeutic strategy. Nevertheless, a greater understanding of the interactions between cancer cells and bacteria will definitely be useful for the development of novel anti-cancer management.Interestingly, accumulating evidence indicates a potential role for  in 1868 , the exp in 1868 ,155,156.d azurin ,158,159. enzymes   to evade the barriers/attacks from the host’s immunosurveillance mechanisms in order to survive and grow. In this process, a dynamic tumor microenvironment is created wherein active interactions occur between different biological constituents, e.g., cancer cells, immune cells, various stromal cells, extracellular matrix, blood vessels, and even the nervous system . HoweverP. aeruginosa and Clostridium difficile, apart from fungal and viral infections [Regarding immunomodulatory therapy against cancer, vaccines are currently available for protection against human papillomavirus (HPV) and hepatitis B virus (HBV); HPV is primarily linked with cervical cancer and HBV infection can initiate liver cancer. Different reports have documented that these two vaccines are generally well tolerated among populations throughout the world ,170,171.fections ,177.As mentioned before, patients with solid tumors or hematological malignancies frequently develop infections. Of note, roughly more than 90% of all cancers are solid tumors. Nevertheless, the increased risk of infections in cancer patients could be due to tumor-related factors or therapy-associated complications, which include neutropenia, damage to biological barriers (such as integument and mucosa), obstruction of physiological passages, patient’s age, nutritional status, as well as surgical procedures and instrumentations for diagnosis or therapy . In geneE. coli, Klebsiella spp., P. aeruginosa, viridans group streptococci, and coagulase-negative staphylococci [Although the use of antibiotics is intimately connected with cancer care, an alarming rise in infections with multidrug-resistant bacteria and the scarcity of suitable antimicrobial agents are important problems in the treatment of cancer patients. Among patients with cancer, isolated drug-resistant bacteria include ylococci . It may ylococci . Consideylococci .Staphylococcus aureus and multidrug-resistant P. aeruginosa infections. In addition, the integration of the antimicrobial stewardship program (ASP) and facility of infectious disease consultations (IDC) decreased the use of carbapenem (a β-lactam class antibiotics) without negative patient outcomes. Finally, they concluded that the application of their method might support appropriate cancer management. In another study that was conducted during 2009–2017 in a cancer department in Spain, the investigators noticed that the combination of IDC and ASP benefited antibiotic use in cancer patients, and this improvement was associated with a decrease in mortality due to bacterial infections [In a recent report from Japan, the investigators had studied the effectiveness of antimicrobial stewardship on 32,202 patients during 2018–2021 at a cancer hospital . They obfections . Likewisfections . InteresKlebsiella spp., E. coli, and P. aeruginosa [Among cancer patients receiving chemotherapy, febrile neutropenia is a serious complication and is negatively associated with overall prognosis, including mortality . It is wruginosa . In addiruginosa .C. difficile or invasive fungal pathogens. Overall, different studies have documented that antibiotic prophylaxis can decrease the episodes of febrile neutropenia and infection-linked deaths in patients receiving chemotherapy [S. aureus in cancer patients [Kouranos and his colleagues reviewed lung cancer cases where the patients were treated with prophylactic antibiotics and they noticed that febrile neutropenia, infections, and the duration of hospitalization were significantly reduced . The autotherapy . Additiopatients .Candida, Aspergillus, and Cryptococcus species are common fungal pathogens that cause invasive infections in immunocompromised conditions, including cancer [Like antibacterial management, the antifungal prophylaxis strategy has been employed in several places to control invasive fungal infections in high-risk hematologic cases such as acute myeloid leukemia and myelodysplastic syndrome ,193,194.g cancer . Furtherg cancer . Neverthg cancer ,197. TheInterestingly, Aitken and his colleagues mentioned a number of antimicrobial stewardship strategies that have been effectively implemented in cancer patients . OverallH. pylori infection, which clearly displays a direct etiological connection to tumorigenesis, the neoplastic role of other bacteria cannot be established so decisively. Moreover, the intricate interactions between specific bacteria and the target cells of our body, as well as the exact nature of different virulence factors, are chiefly unidentified. Oppositely, a number of bacterial products are being evaluated for their utilization as anti-cancer therapeutic agents. Therefore, on the one hand certain virulence factors are pathognomonic for the disease course, but on the other hand these biomolecules could play a key role in treatment strategies. While research continues, clinicians should be ever diligent in having patients participate in screening testing, especially those who are at risk for high-virulence pathogens. By eliminating these pathogens early in a disease process, cancer management can be improved. Secondary bacterial infections among cancer patients have a major impact on the overall prognosis and patients’ survival due to the immunosuppressive nature of malignancy and related chemotherapy/radiotherapy. Moreover, along with bacterial infections, an alarming increase in antibiotic resistance is a great challenge for patients’ wellness. Currently, the careful use of antibiotics is the sole method to provide the relief of cancer patients.Many bacteria are linked with the pathological processes of cancer in diverse ways. Apart from


Eupatorium adenophorum and native plant Eupatorium lindleyanum were selected and cultured under intraspecific (Intra-) competition and interspecific (Inter-) competition conditions, involving inoculation with (M+) and without AMF (M−) and three different levels of P addition including no addition (P0), addition with 15 mg P kg−1 soil (P15), and addition with 25 mg P kg−1 soil (P25) for the two species. Root traits of the two species were analyzed to study the response of the two species’ roots to AMF inoculation and P addition. The results showed that AMF significantly promoted the root biomass, length, surface area, volume, tips, branching points, and carbon (C), nitrogen (N), and P accumulation of the two species. Under M+ treatment, the Inter- competition decreased the root growth and nutrient accumulation of invasive E. adenophorum but increased the root growth and nutrient accumulation of native E. lindleyanum relative to the Intra- competition. Meanwhile, the exotic and native plants responded differently to P addition, exhibiting root growth and nutrient accumulation of invasive E. adenophorum increased with P addition, whereas native E. lindleyanum reduced with P addition. Further, the root growth and nutrition accumulation of native E. lindleyanum were higher than invasive E. adenophorum under Inter- competition. In conclusion, exogenous P addition promoted the invasive plant but reduced the native plant in root growth and nutrient accumulation regulated by AMF, although the native plant outcompeted the invasive plant when the two species competed. The findings provide a critical perspective that the anthropogenic P fertilizer addition might potentially contribute to the successful invasion of exotic plants.Plant invasion has severely damaged ecosystem stability and species diversity worldwide. The cooperation between arbuscular mycorrhizal fungi (AMF) and plant roots is often affected by changes in the external environment. Exogenous phosphorus (P) addition can alter the root absorption of soil resources, thus regulating the root growth and development of exotic and native plants. However, it remains unclear how exogenous P addition regulates the root growth and development of exotic and native plants mediated by AMF, affecting the exotic plant invasion. In this experiment, the invasive plant  Heracleum sosnowskyi had more root branches and root hair development than congeneric native H. sibiricum, leading to a higher nutrient absorption efficiency of the invader than native, thus helping the invasive H. sosnowskyi win the competition with the native H.sibiricum. Therefore, root traits play a vital role in the competition between alien and native plants. In invasion ecology, nevertheless, the majority of trait-based comparisons are concentrated on the more accessible above-ground plant traits [Exotic plant invasions can seriously threaten local communities’ species diversity and stability, causing substantial economic losses and ecological damage ,2. An est traits ,12. ThusFlaveria bidentis, resulting in a higher competitive advantage. However, P is difficult for plants to obtain as most soil P is bound to the surface of molecules or minerals and has low mobility, thus limiting plants’ growth [Below-ground root competition between exotic and native plants is primarily impacted by soil nutrient availability changes . General’ growth . Given t’ growth ,18,19,20Bidens biternata by promoting P absorption, helping natives to resist the alien Bidens alba. Additionally, Sun, Yang [Microstegium vimineum and increase P absorption capacity, thus helping invaders to invade successfully [Arbuscular mycorrhizal fungi (AMF) are a class of soil microorganisms that have long been in a mutually beneficial symbiotic partnership with plants and have been extensively studied, including invasion ecology ,22. AM fun, Yang  concludeessfully . Additioessfully . HoweverEupatorium adenophorum, and a native species, Eupatorium lindleyanum. The aim was to explore how external P addition altered the root morphological characteristics of the two plants regulated by AMF, thus affecting the invasion of alien species.Indeed, with increased human activities, such as excessively added P fertilizer, the soil P availability is inordinately altered . The plaE. adenophorum and the native E. lindleyanum showed high root mycorrhizal colonization after (40–76.2%) inoculation with AMF , 0.503 g/kg total nitrogen (TN), 410.576 mg/kg available nitrogen (AN), 0.704 g/kg total phosphorus (TP), 5.023 mg/kg available phosphorus (AP), 6.489 g/kg total potassium (TK), and 195.443 mg/kg available potassium (AK). In addition, the experimental fungus inoculum +) and without (M−) fungus Glomus etunicatum for alien plant E. adenophorum and indigenous plant E. lindleyanum; (2) the competition treatments involved intraspecific (Intra-) competition, with two alien plant E. adenophorum or two indigenous plant E. lindleyanum separately planted in a plot, and interspecific (Inter-) competition, with one alien plant E. adenophorum plus one indigenous plant E. lindleyanum mixed planted in a pot; (3) the P addition treatments included three different P additions, involving no P addition (P0), 15 mg/kg P addition (P15), and 25 mg/kg P addition (P25). The amount of P addition in this experiment was determined according to the Chinese soil nutrient classification standard [2PO4) in 200 mL of distilled water and then sprayed it evenly over the soil surface with a spray bottle. In this experiment, we selected 3–5 seeds of invasive E. adenophorum and native E. lindleyanum of similar size, disinfected them with 10% hydrogen peroxide for 10 min, and then washed them under distilled water three times. Thereafter, we put approximately 2 kg of sterilized soil into each sterilized plastic pot and then added 50 g of fungus inoculum evenly into the soil. After germination, only two seedlings with good growth were retained in each plastic pot. The remaining plants received P addition once a week for one month. Ultimately, the plant roots were reaped, and the indexes were determined one month after the completion of the P application. The experiment design included two AMF treatments, three P addition treatments, three planting treatments, and six replicates, totaling 108 samples.A potting experiment was performed using a plastic pot , and each plastic pot had a round hole of 1 cm in diameter at the bottom to avoid water accumulation. The experiment contained three factors: (1) the AMF treatments included inoculation with (Mstandard . SpecifiWe adopted the method of Giovannetti and Mosse  to deter+ and M−), competition (Intra- and Inter-), and P addition  treatments and their interactions on the root mycorrhizal colonization, root biomass, root morphological traits, root C, N, and P accumulation. The significant differences between M+ and M−, Intra- and Inter-, among P0, P15, and P25 treatments on root mycorrhizal colonization, biomass, morphological traits, and C, N, and P accumulation at 0.05 level were determined with the least significant difference (LSD) test. All figures were produced by Origin 2022.The experimental data were analyzed by SPSS 27.0 software, and all data were tested for normality and homogeneity of variance before analysis. We used three-way ANOVA to test the effects of AMF (ME. adenophorum, and the native plant E. lindleyanum decreased with increased P addition. AMF significantly promoted the root growth and nutrient accumulation of the two species. When inoculated with AMF, the interspecific competition decreased the root growth and nutrient accumulation of invasive E. adenophorum but increased the root growth and nutrient accumulation of native E. lindleyanum relative to the intraspecific competition. Meanwhile, the exotic and native plants responded differently to P addition, exhibiting the root growth and nutrient accumulation of invasive E. adenophorum increased with P addition, whereas native E. lindleyanum decreased with P addition, indicating the exotic and native plants responded differently to P addition. Further, the root biomass, length, surface area, volume, average diameter, tips, branching points, and C, N, and P accumulation of native E. lindleyanum were greater than invasive E. adenophorum under interspecific competition. Overall, exogenous P addition promoted the invasive plant but decreased the native plant in root growth and nutrient accumulation regulated by AMF, although the native plant outcompeted the invasive plant when the two species competed. The findings may potentially provide evidence that anthropogenically applied P fertilizer increases the possibility of alien plant invasion, providing a perspective to understand the plant invasion mechanisms.We concluded that exogenous P addition reduced the root mycorrhizal colonization of the exotic plant


Cryptosporidium infection has been best studied in mice, where it is mediated by both innate and adaptive immune responses. Dendritic cells are the key link between innate and adaptive immunity and participate in the defense against Cryptosporidium infection. While the effector mechanism varies, both humans and mice rely on dendritic cells for sensing parasites and restricting infection. Recently, the use of mouse-adapted strains C. parvum and mouse-specific strain C. tyzzeri have provided tractable systems to study the role of dendritic cells in mice against this parasite. In this review, we provide an overview of recent advances in innate immunity acting during infection with Cryptosporidium with a major focus on the role of dendritic cells in the intestinal mucosa. Further work is required to understand the role of dendritic cells in the activation of T cells and to explore associated molecular mechanisms. The identification of Cryptosporidium antigen involved in the activation of Toll-like receptor signaling in dendritic cells during infection is also a matter of future study. The in-depth knowledge of immune responses in cryptosporidiosis will help develop targeted prophylactic and therapeutic interventions.Host immune responses are required for the efficient control of cryptosporidiosis. Immunity against  Cryptosporidium is an intestinal protozoan parasite that causes severe diarrhea in human infants and young ruminants [Cryptosporidium exists, among which Cryptosporidium parvum (C. parvum) and C. hominis are known to be responsible for the most clinically significant infections in humans around the world [C. parvum is zoonotic and infective to a broad host range, including animals and humans, while C. hominis is the primary cause of human-to-human infection [Cryptosporidium undergoes several rounds of asexual followed by sexual replication, which gives rise to new oocysts. Oocysts are shed in high numbers, and they are highly infectious and environmentally stable, leading to transmission [uminants . It is tuminants ,2. Accoruminants ,4. Childhe world . C. parvnfection ,7. Infecnfection ,8. The enfection ,10. Withsmission . The impsmission .Cryptosporidium is an intracellular parasite, but it remains extra-cytoplasmic without resulting in systemic infections [Cryptosporidium infection in mice and humans is mediated by both innate and adaptive immune responses; however, immune responses often differ between mice and humans [Cryptosporidium infection primarily involves the communication between intestinal ECs and specialized innate immune cells. Besides being a physical barrier, ECs also act against C. parvum infection in mice by producing chemokines to attract immune cells at the site of infection [Cryptosporidium infection, and CXCL-8 is detectable in the stool samples of children infected with cryptosporidiosis [fections ,14. Howefections . Cryptosfections ,17. Immud humans ,19. The d humans ,21,22. Anfection . Studiesridiosis ,24. Furtridiosis ,26, incrridiosis ,28, and ridiosis ,30,31. Tridiosis ,32.C. parvum infection is IFN-γ, a key proinflammatory cytokine, and as a consequence, its neutralization or genetic depletion makes mice more susceptible to infection [C. parvum infection activates the STAT-1 pathway in human and mouse intestinal ECs to induce the expression of interferon-stimulated genes (ISGs) that exert several antiparasitic effector functions. One of the early studies using human enterocyte cell lines concluded that IFN-γ can inhibit C. parvum infection, but inhibitory activities in vitro are modest, and the mechanisms responsible for parasite restriction are not clear [C. parvum in mice revealed that intestinal type-I innate lymphoid cells are a major source of early IFN-γ, and IFN-γ-induced STAT1-mediated activity is uniquely required in enterocytes to limit parasite growth [C. parvum crude extract in cryptosporidiosis patients [C. parvum antibody and the absence of oocyst shedding [C. parvum [C. parvum, specifically CD4+ T cells, whose role in the clearance of parasites has been recently confirmed in the natural mouse model C. tyzzeri [C. parvum from the infected intestine via cytotoxicity by lysing the infected intestinal ECs [Cryptosporidium infection is constantly improving [Cryptosporidium, with a major focus on the role of DCs in the intestinal mucosa.The major mediator of resistance to nfection ,34,35. Iot clear . A recene growth . Furthere growth . Studiespatients . The expshedding . Adaptivshedding . Althoug. parvum ,41,42. T tyzzeri . Studies tyzzeri . Human Cinal ECs . Althouginal ECs , suggestmproving ,46. In tDendritic cells were first discovered by Paul Langerhans in 1868, who was a medical student when he described dendritically shaped non-pigmentary cells in the epidermis . After aCryptosporidium replicates within a parasitophorous vacuole near the apical surface of the intestinal epithelium but does not invade deeper into the host cell cytoplasm [C. parvum does not infect laboratory mice well, but immunocompromised mice are susceptible, and they have been used to glean insight into some of the features important in controlling infection. The recruitment of DCs at the site of infection by the chemokines released from infected intestinal ECs is an important early step in the recognition of this parasite  derived from MyD88 KO mice fail to respond to C. parvum infection [C. parvum showed increased susceptibility to infection [C. parvum infection in mice has been reported to increase TLR4 expression, suggesting it plays a role in activating DCs to detect C. parvum and initiate Th1 immune responses to restrict parasite growth [C. parvum infection [C. parvum infection in human biliary ECs known as cholangiocytes [Toll-like receptors are an evolutionarily conserved family of cell receptors that play a vital role in immune responses by recognizing invading pathogens, including parasites. Most TLRs, upon the recognition of pathogen-associated molecular structures, use a common adaptor protein known as myeloid differentiation protein 88 (MyD88), leading to the activation of transcription factors NF-κB and the production of proinflammatory cytokines ,65. The nfection . Consistnfection . C. parve growth . The impnfection ,68. Consgiocytes ,69.Cryptosporidium infection, there are several alternative models that could explain these findings. TLR4 recognizes bacterial outer membrane component lipopolysaccharide (LPS) [C. parvum genome; hence, it does not express LPS or LPS-like molecules. However, parasite antigens such as Toxoplasma gondii (T. gondii) and Plasmodium glycosylphosphatidylinositol (GPI) have also been described as ligands of TLR4 [C. parvum could also act as TLR4 ligands, although none have been definitively shown to act in this manner. The signal might also arise from contaminants in the oocyst preparation, as they come from fecal material that is rich in pathogen-associated molecular patterns (PAMPs). The inclusion of controls such as heat-inactivated oocysts could help resolve this issue in future studies. Infection with C. parvum also results in the increased intestinal permeability of neonatal mice, resulting in a leaky gut, thereby exposing the gut mucosa to commensal bacteria [C. parvum and the induction of downstream signaling pathways in DCs.Despite the above evidence implicating TLR4 in de (LPS) . The enz of TLR4 ,71,72. Hbacteria . Hence, C. parvum infection, DCs function as a first line of defense and play a critical role in activating immune responses. The depletion of DCs in adult mice is accompanied by a significant increase in oocyst shedding and more intestinal pathology [C. parvum infection [C. parvum infection compared with adults due to the fact they have less well-developed populations of cDC1 cells. Intestinal DCs, especially CD103+ CD11b- DCs, are one of the key players in the innate immune control of C. parvum infection in neonatal mice [C. parvum infection is the lesser recruitment of intestinal CD103+ DCs during the first few weeks of life due to weaker chemokine production by intestinal ECs [Cryptosporidium infection during the acute phase via cytokine production, and they are required for the clearance of the parasite in neonatal mice [Cryptosporidium infection [During athology . The adonfection . Neonatatal mice . One of inal ECs . The Fltinal ECs . The devinal ECs ,75. Batftal mice . Upon innfection ,76. C. parvum and transfer them further to DCs for migration [C. parvum could actively invade DCs that extend into the lumen of the intestine and be transported to mesenteric LNs [C. parvum antigens. This process could potentially occur from the direct parasite capture by transepithelial dendrites, the capture of apoptotic infected intestinal ECs containing parasites, or antigens secreted across the parasitophorous vacuolar membrane followed by DC uptake. Infection studies using transgenic Cryptosporidium parasites expressing fluorescent reporter proteins combined with live cell imaging could help explore these mechanisms.After recruitment to the site of infection, DCs are known to capture antigens, process them, and then migrate to secondary lymphoid organs via the circulatory system to present the antigens to T cells and initiate an adaptive immune response ,78. One igration . DCs preigration . Furthereric LNs . A previeric LNs . It is nC. parvum infection [C. parvum sporozoite and recombinant antigens have been shown to not only activate murine DCs but also human DCs to produce cytokines [C. parvum infection, IL-12 is a key cytokine involved in promoting Th1 responses [C. parvum infection greatly limits infection severity [C. parvum infection in an IFN-γ-dependent manner [Dendritic cells secrete numerous cytokines, including IL-1β, IL-6, IL-12, IL-18, and TNF-α, upon nfection . Both C.ytokines . Additioytokines . Gut floytokines . During esponses ,84. IL-1severity . IL-12 it manner .Cryptosporidium tyzzeri naturally infects wild and laboratory mice, although different variants cause persistent [C. tyzzeri named Ct-STL, which is a normal part of the endogenous flora of laboratory mice as a non-pathogenic commensal that establishes a long-term infection and only presents as a pathogen in cDC1-deficient mice [Citrobacter rodentium infection and more susceptibility to DSS-mediated colitis in mice [Cryptosporidium might alter responses to other intestinal infections or agents that trigger inflammation. The Ct-STL strain has uncovered the important role of dendritic cells during Cryptosporidium infection, and it should be a valuable tool for future studies. A similar phenomenon has been described following chronic T. gondii infection [rsistent  vs. shorrsistent . Using trsistent ,85. IFN-rsistent . This woent mice . The Ct- in mice . These fnfection , althougCryptosporidium infection than adult mice [C. parvum at an early age were found to be more susceptible to Salmonella infection than adults, thus raising the possibility that altered microbiota may play a role in the detrimental effect of cryptosporidium infection early in life [Cryptosporidium infection and microbial dysbiosis [Cryptosporidium-associated diarrheal symptoms at the time of infection [Cryptosporidium-associated diarrhea, while a higher abundance is associated with subclinical infection [Firmicutes genus and classified within the class Clostridia. Megasphaera is known to synthesize short-chain fatty acids (SCFAs), which help maintain the homeostasis of gut health [C. parvum [C. parvum growth, while long-chain unsaturated fatty acids enhance parasite invasion [Cryptosporidium infection impacts the intestinal microbiota composition, resulting in an increased prevalence of bacterial communities belonging to the Phylum Bacteroidetes [Cryptosporidium infection in early life.The microbiota plays a critical role in the training and development of intestinal immunity . Neonatault mice . The difult mice ,89. The ult mice ,91. Cons in life . Studiesysbiosis ,94,95. It health ,97. SCFA. parvum . Interesroidetes . During roidetes . A recenroidetes ,101. HenC. parvum and mouse-specific C. tyzzeri have provided tractable systems to study the host immune response to this parasite. C. parvum does not infect laboratory mice well; in contrast, C. tyzzeri naturally infects wild and laboratory mice, although different variants cause persistent [Cryptosporidium will greatly help to further investigate host–pathogen interactions. DCs contribute to the host immune response against Cryptosporidium by stimulating innate and adaptive immune mechanisms. Studies have shown DCs as natural adjuvants in different infectious disease models to stimulate the immune response against various types of pathogens [Cryptosporidium.The use of mouse-adapted strains rsistent  vs. shorrsistent . Using trsistent ,103,104 athogens . Clinicaathogens ,107. The


Buddleja (Scrophulariaceae). However, the relationship between macroscopic bark appearance and its microscopic structure remains obscure, hampering the use and interpretation of bark traits in plant taxonomy and phylogenetics as well as in other fields of botany. We studied micro- and macrostructure of bark in the species of Buddleja representing wide taxonomic and geographic diversity to identify general relationships between bark anatomy and morphology. We also examined Buddleja xylem and discussed the importance of anatomical traits for understanding the relationships between clades in this genus. The smooth bark surface in sect. Gomphostigma and the outgroup (Freylinia spp.) relates to the small number of periderms of superficial origin and limited sclerification. This allows for the retention of visible lenticels. In the rest of Buddleja, bark sloughs off and division of labour is present: collapsed phloem undergoes sclerification and acts as a protective layer, while thin-walled phellem forms the separation layers. A similar pattern is found in some groups (e.g. Lonicera), but in others , the pattern is inversed. Wood and bark anatomy supports a sister relationship between the southern African section Gomphostigma and the rest of Buddleja but is taxonomically uninformative among remaining clades. Limited development of periderms and sclerification allows for the retention of a smooth bark surface and conspicuous lenticels. Sloughing off of bark requires division of labour into a lignified protective layer and a thin-walled separation layer. These two functions are never served by a single tissue but are rather divided between phloem and periderm. How more subtle features (e.g. size and shape of fissures) are determined requires further study. Simultaneously, bark anatomy could be a useful source of data to complement molecular phylogenetic studies in a total evidence approach for systematics.Bark  has been extensively studied in recent years, especially its anatomy and physiology. Macromorphological bark characters can be important taxonomically for many plant groups, including the genus  We explored the relationship between bark surface appearance and its microscopic structure using butterfly bushes  as models. Smooth stems with visible lenticles develop when periderms are few, originate superficially, and cells undergo a limited sclerification. Barks that slough off always show division of labour at the anatomical level: some cells become sclerified and protective, others remain thin-walled and act as separation layers. These functions are never served by one tissue. We tentatively linked the anatomy and morphology of bark, but much work is still required to fully understand how other traits (like size of fissures) develop in this plant-environment frontline. Buddleja (Scrophulariaceae) as a model to study the relationship between morphological and anatomical structural levels.Bark anatomy and physiology have recently received much attention . Among tBuddleja consists of 108 woody species (Buddleja (66 species) and Asian sect. Alternifoliae (24 species); eight species are Malagasy , and the rest is from southern Africa (Buddleja: (1) monospecific sect. Salviifoliae , (2) sect. Gomphostigma (with two species: B. virgata and B. incompta), (3) sect. Chilianthus (with four species), (4) monospecific sect. Pulchellae  and (5) Buddleja glomerata of uncertain position (most likely in sect. Chilianthus). The sister position to the rest of Buddleja is not fully resolved and occupied either by sect. Salviifoliae or sect. Gomphostigma . The examined barks vary considerably  or sclerification is only partial (Freylinia). In the remaining species of Buddleja, the first phellogen originates in the outer phloem and subsequent periderms cut off portions of phloem that undergoes solid sclerification; no phelloderm was observed. The similarity of bark structure between Buddleja virgata and Freylinia supports a sister position of sect. Gomphostigma with respect to the rest of Buddleja. The lack of phelloderm in all Buddleja species (except B. virgata) is particularly noteworthy as it implies a shift of cork cambium activity from bifacial to unifacial and would make a rare finding if confirmed  were sampled: 14 species (17 specimens) were obtained from plants cultivated in the National Buddleja Collection at Longstock Park Nursery , one specimen of B. incompta came from a natural population , and a sample of B. saligna was obtained from a cultivated tree . Fourteen species were studied for the first time, two were re-examined (Sixteen species of Gomphostigma (B. incompta), one species from sect. Chilianthus , three species from the New World sect. Buddleja, nine representatives of Asian sect. Alternifoliae, and two hybrids. Herbarium vouchers were deposited in JRAU, NBG or WA  were embedded in GMA, sectioned on an ultramicrotome, stained with toluidine blue, and mounted in Entellan . A universally accepted terminology of bark morphology is missing—we followed the most comprehensive nomenclature system presented by Junnika (1994).Freylinia) and all sections within Buddleja except for sect. Nicodemia and sect. Pulchellae (available samples were too young): we included almost all specimens sampled for the present study  and most specimens examined anatomically by In total, we studied 26 species (32 specimens) from tribe Teedieae , B. incompta (KF027), a thick stem of B. virgata (KF011) and Freylinia lanceolata (KF007) it is homogenous. In thin stems of B. forrestii (KF040) and B. tubiflora , bark texture is also homogenous—likely because multiple periderms have not yet developed. In a thinner stem of B. virgata (KF010), the stem bark is rather homogenous and crumbles only slightly when slashing.Bark texture is loose (crumbling when slashed) in most species. In B. albiflora (KF033), B. dysophylla (KF009), B. crispa (KF042), B. colvilei (KF041) and B. saligna (KF002), which have distinct ripple marks, and B. paniculata (KF036), B. salviifolia (KF013) and B. x weyeriana (KF038), where ripples are indistinct.Bark patterns  are missing, except in B. crotonoides (KF043) and B. tubiflora (KF045), fissures are macroscopically absent but observed under binoculars (possibly due to the specimens’ limited secondary growth). In four species, they are absent or very rare: in B. lindleyana (KF015), the stem is smooth; in F. lanceolata (KF007), B. incompta (KF027) and a thick stem of B. virgata (KF011), the surface is only ruffled . A thinner stem of B. virgata (KF010) resembles B. incompta with slight fissuring/ruffling (the thicker stem of B. virgata [KF011] has a much smoother appearance).Fissuring is present in most species. In B. albiflora [KF033], B. aromatica [KF014], B. auriculata , B. forrestii [KF034], B. globosa [KF032], B. saligna [KF002], B. salviifolia  and possibly also in B. crispa [KF046]); (2) parallel to boat shaped ; (3) parallel to oblique ; (4) wavy (B. coriacea [KF016]); (5) irregular ; or (6) compound (F. tropica [KF008]).Fissuring is of six types: (1) boat shaped (in F. lanceolata (KF007), B. incompta (KF027), B. virgata , B. lindleyana (KF015), B. crotonoides (KF043), B. tubiflora (KF045); flattened in all remaining species except B. cordata (KF005), B. cf. curviflora (KF044), B. coriacea (KF016), B. glomerata  where they are reticulate.Ridges are absent in species without fissuring: Freylinia , the thick stem of B. virgata (KF011), B. incompta (KF027), B. crotonoides (KF043) and B. tubiflora (KF045). In the remaining species, flakes are scaly  or irregular. In B. auriculata (KF012) and B. salviifolia (KF013), it is loose-hanged at the same time or even stringy.Scaling is absent in F. lanceolata (KF007) and B. virgata (KF011), where the bark is lenticellate with lenticels round, scarce, solitary and medium in size. Additionally, the bark surface can be rough (B. cordata [KF005]), smooth , or rugose .External markings are well seen only in The epidermis is composed of a single layer of bottle- to dome-like cells, (16–)25(–34) µm in tangential size with thin walls and prominent cuticle . TrichomThe cortex is composed of ≤10 cell layers: the outermost layer, directly adjoining the epidermis, consists of isodiametric cells; it is followed by a few layers of radially elongated, palisade cells with large, intercellular spaces, and the innermost layers are made up of isodiametric, rounded cells (17–)27(–37) µm in tangential diameter . The perThe first-formed periderm is initiated in the subepidermal layer of the outer cortex. The subsequent periderms are initiated in secondary phloem, and their arrangement is reticulate. Phellem is non-stratified and consists of ≤10 layers of isodiametric to tangentially elongated phelloid cells  with weaConducting secondary phloem is very homogenous and consists of sieve tubes with companion cells , axial parenchyma, and rays . Sieve tSecondary phloem rays are uni- and biseriate. They are composed of square, procumbent and some upright cells forming multiple rows (≤10) flanked by upright cells, or all types of cells are intermixed throughout the ray body. No crystals, tannin deposits or secretory structure were observed.Dilatation of secondary phloem is affected by tangential stretching of sieve tube members, axial parenchyma cells and ray cells, but no anticlinal divisions were found; possibly the broad intercellular spaces in the outer cortex are also the result of dilatation. No sclerification of any secondary phloem elements was observed.B. albiflora, B. crispa, B. crotonoides, B. fallowiana, B. forrestii, B. globosa, B. myriantha, B. nivea, B. paniculata, B. x wardii and B. x weyeriana. The bark structure in stems with fully developed periderm was observed in all the previous species, as well as in B. cf. curviflora, B. colvilei and B. tubiflora.Details of young stem anatomy before the development of periderm were observed in B. albiflora, B. crotonoides, B. fallowiana, B. forrestii, B. x wardii), tangentially elongated , or intermediate (B. x weyeriana) cells  or trichomes were numerous  cells . The epilbiflora , B. croteyeriana  inset.B. albiflora, ≤10 layers in B. crispa, B. crotonoides, B. fallowiana, B. x wardii, and B. x weyeriana; ≤15 in B. forrestii, c. 10 in B. globosa, c. 6 in B. myriantha, ≤12 in B. nivea, ≤15 in B. paniculata; B. paniculata and B. x wardii; in the remaining species, moderately thick-walled pericyclic fibres are disposed as discrete groups of ≤12 cells or continuous, bi- to pentaseriate ring  of isodiametric to radially elongated  and B. forrestii. Section quality did not allow for examination of the transition zone between conducting and nonconducting phloem in B. fallowiana and B. x wardii. In the remaining species, the transition is sharp, typically marked by periderm initiation. The nonconducting phloem consists of weakly obliterated to heavily collapsed elements, and of the sclerified elements. Zone of phloem sclerification is commonly adjacent to the underlying periderm. B. forrestii, B. globosa (B. x weyeriana (B. x weyeriana) with sclereids in small groups and interrupted 1–2(–3)-seriate bands. Other species  show more abundant  sclerification of nonconducting phloem with the sclereids in continuous bands ≤3 cells in width , axial parenchyma, and rays . Sieve teyeriana ; in the B. nivea , and inu globosa  and B. xeyeriana  show tanin width .B. albiflora .Secondary phloem rays are uni- to tetraseriate, most often bi- to triseriate , G. Ray lbiflora , ≤15 roweyeriana , C, or bB. albiflora, B. myriantha, B. nivea, B. paniculata and B. x weyeriana; in the remaining species dilatation was not observed. In all cases, conductive cells, axial parenchyma, and rays in the portions of secondary phloem separated by periderms undergo sclerification  or distinct, marked by tangential rows of radially flattened fibres .Wood is diffuse porous in  species . Growth B. incompta and B. myriantha; B. myriantha where they are more rounded; narrow to very narrow [(16–)19–38(–50) µm wide] and numerous [(112–)125–375(–496) vessels mm2] in all species except for B. incompta, where they are very numerous (mean 955 vessels mm-2). Vessels are partly solitary . They are disposed in small clusters and radial multiples usually of 2–4. There is no distinct pattern of vessel disposition, except for a weak tendency towards diagonal pattern in latewood of B. albiflora , small to medium [(3.2–)4.2–6.9(–7.1) µm in vertical and (4.4–)4.6–8.2(–8.5) µm in horizontal diameters]. Apertures are slit-like (most species), lens-like  or short and almost circular . Vessel–ray pits are similar in size and shape to intervessel pits, except for B. incompta where pitting is often scalariform or gash-like. Vessel-ray pits have clearly reduced borders (pitting in B. paniculata and B. x weyeriana could not be clearly observed). Very fine helical thickenings are present in most vessels and throughout their length in all species except B. x wardii (mostly in narrower vessels) and for B. incompta (completely missing). Numerous vascular tracheids are present in B. albiflora, B. forrestii, and B. colvilei, while fibriform vessels 246–435(–1256) µm long. Perforation plates are exclusively simple. Intervessel pitting is alternate with pits mostly circular and rounded, sometimes polygonal (often in s (sensu ) were obB. cf. curvifora and B. paniculata), and thin- to thick-walled with simple to minutely bordered pits (B. paniculata and B. x weyeriana). In B. incompta, numerous parenchymatic cells observed in tangential longitudinal sections may either be solitary axial parenchyma cells or small, uniseriate rays . Uniseriate rays are composed of square and upright cells in variable proportions. Multiseriate rays consist of rows of procumbent cells  sometimes mixed with square cells and flanked by 1–4 rows of square and upright cells; of square, upright, and procumbent cells intermixed throughout the ray body ; or mostly of square and upright cells intermixed with rare procumbent cells .Rays are uni-, bi- to tetraseriate . Mean nuB. globosa and B. tubiflora contained prismatic crystals, and in ray cells of B. fallowiana, B. forrestii, B. nivea, B. paniculata and B. x wardii droplets of tannins were found.Upright and square, rarely also procumbent, ray cells of Buddleja, section Gomphostigma is like the sister group of the genus (Freylinia) in bark appearance and anatomy, and dissimilar to all remaining sections. Their species have a smooth, non-peeling, little or non-fissured bark surface, and homogenous texture. Instead, other Buddleja species share distinctly fissured and peeling bark with loose texture , Melaleuca (Myrtaceae), Dodonaea (Sapindaceae) (Vitis (Vitaceae)  with stringy bark  . Howeveritaceae)  and in Engy bark , the divngy bark .Lonicera, most Buddleja; Vitis). Theoretically two other modes are possible: (3) both functions can be performed exclusively by secondary phloem, or (4) only by periderm. We did not find, however, any data supporting the existence of these options.Thus, stringy/scaly barks of similar appearance can arise from two different microstructural ground plans: either with (1) periderms (phellem) acting as separation layers combined with protective secondary phloem . Also, the lenticels found in F. lanceolata and B. virgata are common in species with non-scaly barks , and cut-off phloem undergoes sclerification—a feature not reported outside Buddleja  were treated in the subfamily Buddleioideae within Loganiaceae. Solereder listed three diagnostic characters for Buddleioideae: (1) lack of intraxylary phloem, (2) presence of branched and glandular trichomes, and (3) pericyclic origin of the first periderm. The first feature differentiated Buddleioideae and Loganioideae – a group of species now known to be distantly related to Buddleja – and we can confirm that intraxylary phloem never occurs in Buddleja, although it is rarely found in other Scrophulariaceae (Scrophularia) . This feature was pointed out by Neither Solereder nor other early authors  mentioneFreylinia (Scrophularia (Verbascum (S. latifolia) to numerous, thick-walled sclerified cells (e.g. V. arcturus). Nevertheless, none of the examined species undergoes nearly solid phloem sclerification as reported in most Buddleja species, and the most similar condition is found in F. tropica (but not in F. lanceolata). Conversely, three of nine Scrophularia species and seven of nine Verbascum species in the Xylem Database  and Verbascum (V. arcturus). In all other studied members of Scrophulariaceae, phellem consists of thin-walled (phelloid) cells, and phloem and ray dilatation occurs commonly.Scrophulariaceae are poorly studied anatomically: most species hitherto examined actually belong in Orobanchaceae or Plantaginaceae  due to its obliteration.Our examination of semi-thin sections of  species , D. It iBuddleja . The two latter species—the sole members of sect. Gomphostigma—are also characterised by a markedly greater number of uniseriate rays mm-1 than in any other Buddleja . These rays are often short and resemble short strands or fusiform solitary cells of axial parenchyma. Both species of sect. Gomphostigma are also distinct in a very high number of vessels mm–2 , which in turn leads to the low percentage of solitary vessels. Consequently, the combination of high vessel density, vessels contacting other vessels, very numerous short and uniseriate rays resembling axial parenchyma, and lack of helical thickenings can distinguish sect. Gomphostigma from the other sections of Buddleja.Scarcity or absence of axial parenchyma is universal among tahensis , B. virgBuddleja sect. Gomphostigma may represent the plesiomorphic state for the genus, since they differ from those observed in the rest of the genus but share many similarities with traits in Freylinia species in tribe Teedieae (the sister to Buddleja). This supports sect. Gomphostigma as sister to the rest of Buddleja. Phylogenetic analyses of high-throughput sequencing of loci from targeted sequence capture resolved sect. Gomphostigma as sister to the rest of the genus with very strong support .Bark—and to a lesser degree wood—anatomy support a sister relationship between South African The following additional information is available in the online version of this articleAppendix A – Accession information for specimens of Buddleja used in the studyAppendix B – Tabulated summary of bark anatomical and morphological traits in Buddleja and Freylinia.plad003_suppl_Supplementary_Appendix_AClick here for additional data file.plad003_suppl_Supplementary_Appendix_BClick here for additional data file.