Babesia parasites that can cause severe disease and death. While blood type is known to affect the mortality of Plasmodium falciparum malaria patients, associations between red blood cell (RBC) antigens and Babesia microti infection and disease severity are lacking.Babesiosis is an emerging infectious disease caused by intraerythrocytic Babesia-infected (18S rRNA reactive) blood donors in 10 endemic states in the Northeastern and northern Midwestern United States. We also assessed possible associations between RhD and ABO blood types and disease severity among hospitalized babesiosis patients in Connecticut.We evaluated RhD and ABO blood types of Babesia-infected blood donors were analyzed, of which 750 (97.7%) had detectable B. microti-specific antibodies. B. microti-infected blood donors were more likely to be RhD-  than RhD+ donors. Hospitalized RhD- babesiosis patients were more likely than RhD+ patients to have high peak parasitemia , which is a marker for disease severity. No differences in RhD+ blood type were noted between residents of the Northeast  and the Midwest . Overall, ABO blood type was not associated with blood donor B. microti infection, however, B. microti-infected donors in Maine and New Jersey were more likely to be blood type B compared to non-type B , while infected donors from Pennsylvania were less likely to be type B compared to non-type B (OR 0.32 [p = 0.02]).A total of 768 B. microti infection and babesiosis severity. The association of B antigen with B. microti infection is less clear because the antigen appeared to be less prevalent in infected Pennsylvania blood donors but more prevalent in Maine and New Jersey infected donors. Future studies should quantify associations between B. microti genotypes, RBC antigens, and the frequency and severity of B. microti infection to increase our understanding of human Babesia pathogenesis and improve antibody, vaccine, and RBC exchange transfusion strategies.People expressing RhD antigen may have a decreased risk of  Babesia microti infection and both red blood cell RhD and B antigens. Numerous studies have quantified the impact of ABO blood type on Plasmodium falciparum (Pf) infection, an intraerythrocytic pathogen in the same Apicomplexa phylum as Babesia. For example, type O blood has been noted to protect against severe malaria, which may explain why type O is prevalent in Pf-endemic regions. Studies of ABO and RhD blood types and B. microti infection are lacking. The American Red Cross has a large blood donor dataset from B. microti-endemic states that can be used to study how ABO and RhD blood types are linked to B. microti infection, as measured by the presence of B. microti 18S rRNA. Data from 10 B. microti-endemic states showed that B. microti-infected blood donors were significantly less likely to be RhD+  than RhD- donors. A review of babesiosis patients admitted to Yale New Haven Hospital between 2011 and 2021 revealed that RhD+ patients were significantly less likely than RhD- patients to have a high (>5%) peak parasitemia (p = 0.017), which correlates with disease severity. Thus, the RhD antigen on the RBC membrane may decrease the risk of B. microti infection and disease severity. The blood type B antigen may affect the frequency of B. microti infection. The odds ratios (ORs) of B. microti infection in blood donors for blood type B compared to non-type B were 2.49 (p = 0.008) in Maine and 2.07 (p = 0.009) in New Jersey, but 0.32 (p = 0.02) in Pennsylvania. Future studies should quantify the impact of RhD, ABO, and other RBC antigens on Babesia infection, and the mechanisms by which they impact RBC invasion and babesiosis severity.We found an association between  Babesia, which are unicellular intraerythrocytic organisms. Almost all cases in the US are caused by Babesia microti and primarily transmitted to humans by Ixodes scapularis ticks. However, Babesia can also be acquired via blood transfusion, perinatal transmission, and organ transplantation  and 2.07 [p = 0.009]), respectively  admission were evaluated. Peak parasitemia was defined as the highest percentage of RBCs containing Babesia parasites identified on peripheral blood smears during hospitalization. A total of 120 patients were evaluated with the following frequency of blood types: A (n = 47), B (n = 11), AB (n = 6) and O (n = 56). The majority of patients were RhD+  and the rest were RhD- .Blood types and RhD+ patients were less likely  to have high parasitemia levels of >5% . There wBabesia-infected than donors without the antigen (RhD-). The odds ratio for RhD+ blood donors in the Midwest and Northeast were both less than 1.0, despite differences in B. microti genotypes in these two regions [B. microti entrance and/or proliferation within RBCs. We found that ABO blood type generally does not correlate with Babesia infection or disease, although type B blood donors from Maine and New Jersey were more likely to be Babesia-infected while those from Pennsylvania were less likely to be Babesia-infected.We found that blood donors who have the RhD RBC antigen (RhD+) are less likely to be  regions . Among h regions . Thus, dBabesia infection and/or disease is unknown. Studies comparing RhD+ and RhD- individuals with Plasmodium infection in Africa have yielded inconsistent findings with some studies showing RhD+ individuals more likely to be Plasmodium infected and other studies showing RhD+ individuals less likely to be Plasmodium infected [Plasmodium species or genotypes–which were not always accounted for. In one study, Pf-parasites and RBCs of four different blood groups were co-cultured. Parasite invasion was less for type B RBCs than for type A and type O RBCs. This appeared to be due, at least in part, to the amount of surface H antigen in each blood group [Babesia strains have different surface antigens that may bind more or less strongly to B antigens on RBCs. It has been shown that inland and coastal Babesia genotypes in Massachusetts differ [Babesia surface antigens may explain why type B blood donors in Maine and New Jersey  were more likely to be Babesia-infected and those from Pennsylvania (an inland state) were less likely to be infected. Thus, the RBC B antigen may promote invasion for some B. microti genotypes while resisting invasion for other genotypes.The mechanism(s) of RBC antigen enhancement or inhibition of infected –25. Diffod group . Differes differ . DiffereEstablishing a connection between blood type and an infectious disease can be problematic, as illustrated by the often-discrepant findings of research on ABO blood group and SARS-CoV-2 infection and COVID-19 severity. Differing findings may be due, at least in part, to viral genetic variants, which were not always identified in these studies –31. TherBabesia species or genotypes. The Babesia assay used to test blood donors does not distinguish between B. microti, B. divergens, B. duncani, and B. venatorum, however, B. microti is the only Babesia species found at our study sites. In addition, 97.7% of Babesia-reactive blood donors had B. microti-specific antibodies. The only Babesia species that is endemic in Connecticut is B. microti. The blood-donor analysis did not include percent parasitemia, and blood donors are not exactly representative of the general population. For example, blood donors in the US tend to be healthier than the general population [B. microti infection were only found in three states and Pennsylvania had an opposite OR compared to New Jersey and Maine. It is possible that the B antigen does not impact RBC invasion and that B. microti genotypes do not differ in inland Pennsylvania and coastal New Jersey and Maine. Only 12 of the hospitalized babesiosis patients were RhD- blood type. Finally, more comprehensive RBC genotype and phenotype data could have great value because antigens other than ABO(H) and RhD may be important for parasite invasion and disease severity.Our study has several limitations. The ARC and Yale New Haven Hospital data do not include information about the pulation , and minpulation . StatistBabesia infection and severe babesiosis may help improve the treatment and prevention of babesiosis. Current antibiotic therapy is effective in most cases but inducible antibiotic resistance can develop in B. microti during prolonged duration with standard antibiotic regimens in immunocompromised hosts [Babesia entrance into RBCs might help in the development of antibiotics that impair parasite invasion. RBC exchange transfusion is an adjunct therapy used in severe cases of babesiosis [Babesia-resistant RBCs might improve the efficacy of exchange transfusion. For example, M- and/or S-antigen negative RBCs show resistance to B. divergens invasion in vitro and have been recently proposed for use in RBC exchange transfusion for the treatment of this type of babesiosis [B. divergens mortality can exceed 40% in immunocompromised patients and rebound parasitemia can occur after RBC exchange transfusion. Similarly, because type O RBCs offer protection against severe Pf malaria, due to decreased rosetting and cytoadherence, they have been proposed for use in RBC exchange transfusion [Identifying the mechanisms of RBC blood type risk factors for the development of ed hosts –39. A bebesiosis ,40. Use besiosis . B. divensfusion .Babesia acquisition and babesiosis mortality are needed. For example, narrowly focused genotyping of the Babesia strains isolated from the Babesia-reactive type-B blood donors in Pennsylvania, New Jersey, and Maine might be an efficient way to identify different Babesia antigens and genotypes, revealing pathogen diversity that might promote development of effective monoclonal antibody combinations and/or a human Babesia vaccine.Additional studies that clarify possible associations between blood types and S1 Fighttps://commons.wikimedia.org/wiki/File:Usa-state-boundaries-lower48%2B2.png.The association was not statistically significant using a chi-square test. This map was cropped from a public domain version created by Brian Szymanski found at Wikimedia Commons website (TIFF)Click here for additional data file.S2 Fighttps://commons.wikimedia.org/wiki/File:Usa-state-boundaries-lower48%2B2.png.The association was not statistically significant using a chi-square test. This map was cropped from a public domain version created by Brian Szymanski found at Wikimedia Commons website (TIFF)Click here for additional data file.S3 Fighttps://commons.wikimedia.org/wiki/File:Usa-state-boundaries-lower48%2B2.png.The association was not statistically significant using a chi-square test. This map was cropped from a public domain version created by Brian Szymanski found at Wikimedia Commons website (TIFF)Click here for additional data file.


Pseudogymnoascus destructans in China. In this study, we characterized the skin microbiomes of four bat species at five localities using 16S rRNA gene amplicon sequencing to understand their skin microbial composition, structure, and putative relationship with disease. The alpha- and beta-diversities of skin microbiota differed significantly among the bat species, and the differences were affected by environmental temperature, sampling sites, and host body condition. The chiropteran skin microbial communities were enriched in bacterial taxa that had low relative abundances in the environment. Most of the potential functions of skin microbiota in bat species were associated with metabolism. Focusing on their functions of defense against pathogens, we found that skin microbiota could metabolize a variety of active substances that could be potentially used to fight P. destructans. The skin microbial communities of bats in China are related to the environment and the bat host, and may be involved in the host’s defense against pathogens.Host-associated skin bacteria are essential for resisting pathogen infections and maintaining health. However, we have little understanding of how chiropteran skin microbiota are distributed among bat species and their habitats, or of their putative roles in defending against  Animal skin is the first line of defense against pathogen invasion and carries diverse symbiotic microbiota that affect host development, reproduction, and health . MeanwhiThe host species is generally a valuable predictor of skin microbiome structure . In 38 mEnvironment is another important factor influencing skin microbial community structure due to continuous skin contact with the external environment . MassiveBatrachochytrium dendrobatidis disturbed the amphibian skin bacterial community composition and structure in a natural epidemic and in experimental infections  in China  predict and compare microbiome putative functions, with an emphasis on those related to pathogen defense.We investigated the skin microbiome of four widely distributed bat species across five localities in China during late hibernation, when bats typically have the greatest fungal load and prevalence . We atteMu. leucogaster (n = 46), R. ferrumequinum (n = 59), Myotis petax (n = 17), and Rhinolophus pusillus (n = 9). We also collected corresponding environment samples (n = 35) from five localities: New cave (Jilin Province), Gezi cave (Jilin Province), Di cave (Jilin Province), Temple cave (Liaoning Province), and Water channel (Henan Province). Data was collected at the end of March and the beginning of April in 2018  five times using linear strokes. These samples were stored in 500 μl RNAlater  tubes at −20°C within 24 h of sampling until returning to the laboratory, where samples were stored in −80°C refrigerator for DNA extraction. We measured bat roosting temperatures using an infrared thermometer . After swabbing, we measured bat forearm length and weight and calculated the body mass index (BMI = weight/forearm length) and identified the sex and species. Bats were released immediately after sampling. Sampling was performed by the same person to limit individual differences in swabbing techniques. All studies were approved by the Laboratory Animal Welfare and Ethics Committee of Jilin Agricultural University.We dipped sterile polyester swabs in sterile water and wiped five times along the bats’ forearms and muzzles, as previously described , to testP. destructans ATCC MYA-4855 isolate (P. destructans following the method of P. destructans load = 10^[(Ct −22.04942)/−3.34789] . The modified protocol included the use of lyticase during the lysis step . Each DN isolate . We used3.34789] .Genomic DNA was extracted from swabs using the E.Z.N.A™ Mag-Bind Soil DNA Kit  according to manufacturer protocol. PCR amplification was used on the first pair of primers  to target the V3–V4 region of the 16S rDNA and contained 17 bp barcodes . The PCRQ ≥ 20). After quality processing, a total of 6,705,497  and 1,940,539 reads  were obtained from bat and environment samples, respectively. Sequences were processed using VSEARCH v. 7.0.1234 ], and the number of reads in the environment samples was 2,043,422 . We used Cutadpat to remove adapter sequences from the high-throughput sequencing reads . Quantit7.0.1234  and then7.0.1234 . The OTU7.0.1234 . The OTUT-tests and Wilcoxon tests were used to compare alpha diversities between host species and their environment samples. Beta diversity was calculated with Bray Curtis distance matrices and plotted using non-metric multidimensional scaling (NMDS) in k = 2 dimensions using the ordinate and plot_ordination functions in the R package phyloseq  of skin microbial diversity of host and environment samples was calculated. phyloseq . Permuta package . Kendal’ package . We usedP. destructans infection status, and P. destructans load in OTU richness and Shannon diversity. The calculation and visualization of beta diversity among host species and sites was as described above. To assess the influence of each explanatory variable  on host species microbiota composition, we used partial distance-based redundancy analysis (db-RDA) while controlling for the host and site variables. Adjusted R-squared (R2) values were calculated to clarify the explanatory variables (anova.cca function of the vegan package. The unweighted pair group method with arithmetic mean (UPGMA) was used on Bray Curtis distances of mean >0.1% for OTU relative abundances at the genus level to determine clustering patterns across host species. We performed generalized linear mixed models (GLMMs) to assess OTUs that were differentially abundant between species in the glmmTMB package using a beta distribution and a logit link  with a quasi-Poisson distribution to examine variation depending on factors such as sites, BMI, temperature, sex, ariables . Signifigit link . In thisFunctional prediction was conducted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States2  and beta-diversities across four sites. We used indicator Value tests    to identfunction , and sigdicators . Kendal’on 4.0.5 .P < 0.001). Myotis petax had the highest P. destructans infection intensity (average log-transformed P. destructans loads: −2.62), followed by R. ferrumequinum (−4.20), Mu. leucogaster (−4.24), and R. pusillus (−6.11)  , and the highest P. destructans prevalence was in Gezi cave (−3.69)  . The higM. petax . Among d (−3.69) .R. pusillus , but similar in R. ferrumequinum  and R. pusillus  , but similar in R. ferrumequinum  and R. pusillus   . The Sha= 0.145) . All hosMu. leucogaster having the highest correlation . When we calculated ranked correlations with the most relatively abundant OTUs (OTUs > 0.3%), all of the correlations between bat and environment samples became significantly negative   .M. petax to 5.8% for R. ferrumequinum)  and BMI  were the significant factors associated with changes in OTU richness. Temperature, sex, P. destructans infection status, and fungal load did not significantly affect alpha diversity measures . Shannon diversity was also significantly different from host species across all sites  and BMI  were the significant factors associated with changes in Shannon diversity.Host species shared the majority of the relative abundances of OTUs on skin bacterial communities in all four species (45.6%). Few OTUs unique to individual host species were found (ranging from 0.4% for equinum) . OTU ricF3,130 = 3.41, P = 0.001, R2 = 0.074; F3,130 = 4.11, P = 0.001, R2 = 0.115). The partial db-RDA analysis showed that bat roosting temperature was significantly correlated with microbial composition changes  when we controlled for the host species and sites. Likewise, P. destructans infection status, fungal load, BMI, and sex did not significantly affect the beta diversity measures . All 21 OTUs were differentially abundant among the bat species  in gene relative abundances from KEGG Orthology groups (KOs) on chiropteran skin microbiomes using PICRUSt2  was found to significantly affect it (GLM: P > 0.05). Shannon diversity of Mu. leucogaster was not significantly different across sites . The mean relative abundance of bacterial taxa on Mu. leucogaster differed across sites (Pseudomonas was the dominant genus in Gezi cave (36.1%), Temple cave (26.9%), and Di cave (19.4%). However, the dominant genus in New cave was Myroides (19.3%). Multiple indicator taxa were detected at different locations, such as Salinibacterium, Rhodococcus, and Pseudomonas at Gezi cave (P. destructans loads and relative abundance (>0.1%) of many genera on Mu. leucogaster across sites showed a negative correlation, but the results were not statistically significant (data not shown).Operational Taxonomic Unit richness of ss sites . Pseudomezi cave . The P. Mu. leucogaster , and most KOs were associated with metabolism  can be found in the article/The animal study was reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of Jilin Agricultural University.ZL performed data analysis and drafted the manuscript. ZL, AL, WD, HL, SL, and LJ collected the samples. KS and JF developed the study concept, design, and supervised the project. AL and KS reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Agave marmorata Roezl has been classified as an endangered species. Extracting these plants from the forest for commercial purposes and long maturation periods of close to 30 years have contributed to their loss. A. marmorata interacts with pollinators and other Agaves species to maintain genetic variability. Thus, the conservation and restoration of the agave ecosystem is an ecological challenge. Typically, agave micropropagation use meristem or leaves as explants to rapidly produce uniform agave plants in age and size on a large scale leading to homogeneous plantations. However, introducing these clones to the field reduces genetic variability. This study evaluated in vitro micropropagation of A. marmorata from seeds to generate clonal lines. The selected seedlings exhibited variations in multiplication capacity and stable tissue formation. Variations in clonal lines could be exploited to produce high-quality plants with different capacities, such as faster propagation, enhanced stress adaptation, and continued growth under nutrient limitation conditions, consequently maintaining genetic variability. Furthermore, some clonal lines were inoculated with four endophytic bacteria to identify other differences among these plants, including endophyte-host compatibility. Variable responses to inoculation were observed among clonal lines. We found that Achromobacter xylosoxidans was compatible, unlike Enterobacter cloacae which caused plant death.The wild A. marmorata is the raw material used for tepextate mescal production but is classified as an endangered species. In the present study, we obtain and multiply clonal lines of Agave marmorata Roezl by selecting seedlings derived from seeds. Ten seedlings from two lots of 400 germinated seeds were selected for axillary bud proliferation induced by BAP 5 mg/L in vitamin-free Murashige and Skoog’s medium. Differences in shoot numbers, heights and senescent tissue formation were observed. Notably, the AM32 line formed 84 shoots and presented low senescent tissue after 60 d of culture. We also selected the AM31 and AM33 clonal lines. Four-month shoots were extracted with 80% methanol in water to determine the total content of saponins, flavonoids, and phenolic acids and compare the three clonal lines. Some bioactive molecules were identified using HPLC techniques and MALDI-TOF mass spectrometry none showed significant differences in content. Additionally, plants derived from the clonal lines were inoculated with four endophytic bacteria. Among these, Achromobacter xylosoxidans supported plant growth of AM32. A notable effect of plant death was observed after inoculation with Enterobacter cloacae, an endophyte of A. tequilana. Additionally, Pseudomonas aeruginosa, an endophyte from A. marmorata, reduced biomass. Our results demonstrate the incompatibility of A. marmorata to E. cloacae and specialization between the host plant and its endophytes. The compatibility of the plant-endophyte could be exploited to boost the establishment and stability of mutualisms to benefit plant development, stress tolerance and pathogen resistance. The differences in multiplication capacity, stable tissue formation, and endophyte biotization responses may indicate genetic variability. Clonal selection and micropropagation from seed-derived plants could contribute to conserving the endangered A. marmorata plant for reforestation in their natural habitats, thus, assuring mass propagation for sustainable industrial production of mescal, bioactive compounds, and prebiotics. Agave  at m/z 1201), Gallic Acid , Caffeic Acid  and Sinapic Acid , while the flavonoids were represented by Isorhamnethin Rutinoside , Isorhamnetin-Glucuronide , Isorhamnetin 3-Glucoside , Isorhamnetin-3-O-Diglucoside , Myricetin Glucoside , Myricetin 3-O-Glucuronide , Myricetin 3-O-Rutinoside , Rutin , Quercetin-3-O-Diglucoside , Quercetin-3-Glucoside or Isoquercitrin  and Kaempferol Diglucoside .The fractions were analyzed for tentative identification by MALDI-TOF mass spectrometry. Phenolic compounds present in the fractions that absorbed at 275 nm included: Catechin  , AM31 (λm/z 484) and Kaempferol Rutinoside . At 518 nm, fractions contained anthocyanins and hexoses designated as glucose. The AM31 line had the highest Cyanidin 3,5 Diglucoside  content compared to AM32 (NA% 23.08) and AM33 (NA%21.66). Delphinidin 3,5-O-Diglucoside  was slightly higher in the AM31 line. The F18 corresponded to Delphinidin-3-O-Glucoside ([M+Glucoside+Na] at m/z 484) and was slightly elevated in AM32. The HPLC results were generally consistent with the total phytochemical content analyses since the concentrations adjusted to area normalization were similar among clonal lines. Interestingly, at least 30 fractions were absent from the A. marmorata hijuelo leaf extract compared to micropropagated shoots, many being anthocyanins and flavonoid-type compounds.The fractions F25 and F66 (λ = 390 nm) tentatively corresponded to the flavonoids Quercetin-Glucuronide  profiles with slight intensity variations and signal appearances at m/z 1361.978 in the AM33 line  are presented in M33 line C. This mA. marmorata clonal lines. Our intention in applying endophytes was to assess their compatibility and effects on plant growth parameters between clonal lines. We evaluated the effect of an intensive inoculation with four endophytic bacteria on plants of the three selected B. tequilensis and P. aeruginosa. Similarly, no response in new root formation was detected following microbial treatments, except for plants inoculated with A. xylosoxidans (Ax), which formed three more roots than the control. In the AM31 line, E. cloacae inoculation caused plant death. Inoculating AM32 with B. tequilensis (Bt) or A. xylosoxidans (Ax) induced the emergence of one or two new leaves during the post-inoculation period. Control plants did not form leaves. The leaves of plants inoculated with Ax were dark green. The highlighted effect of bacterial inoculation on AM32 is related to the number of roots and their length. For example, Ax increases up to 14 new roots compared to control plants. For the Bt strain, a total of 11 new roots was observed, followed by Pa with 8 and Ec with 3. An effect on root length was associated with the Ax and Pa strains, yielding nine and seven centimeter increases.Ax treatment with 3.82 g. Negative data were observed for Ec, Pa, and Bt with −3.08 g, −2.98 g, and −1.95 g, respectively. For AM31, no significant differences were observed between Ax and Bt (1.32 g and 1.20 g) inoculation in this parameter; however, a negative biomass increment was observed for bacterial treatments with Pa and Ec with −1.40 g and −0.78 g.Although the AM32 line had the most outstanding response to bacterial inoculation, this is not reflected in the total biomass acquisition. The highest dry weight was observed for Bt induced the formation of 10 new roots, with an average close to 11 cm. In the treatments with Ax and Pa, we observed the formation of 5 and 8 roots more than the control, respectively. No significant changes in weight gain were detected. The A. xylosoxidans strain contributed the greatest to root formation in all clonally propagated lines, producing 16 new roots. Here, we found a notable effect on the plant death caused by E. cloacae inoculation and a reduction in biomass acquisition of the AM31 and AM32 lines caused by P. aeruginosa. These observations indicate incompatibility between E. cloacae and the clonal lines and specificity of A. xylosoxidans and P. aeruginosa for A. marmorata.Like the other two clonal lines, endophytic inoculation increased root length and number of AM33 compared to the control. Bacillus tequilensis is an endophytic bacterium contained in the seeds of A. tequilana. It has a good effect on the AM33 line, especially concerning root formation (number and length). In the other clonal lines, its effects were quite limited. Herein, the AM32 line had a moderate response to the inoculation of this bacterium, and some plants died.A. marmorata. Subsequently, the three lines selected for their propagation capabilities were analyzed in terms of their total phytochemical content, with some flavonoids and phenolic compounds identified by mass spectrometry. However, the main difference was the compatibility response presented by the lines to the inoculation of endophytic bacteria native to Agave species, including A. marmorata and A. tequilana.In this paper, we analyzed the germination capacities of more than 400 seeds and the seedlings’ development as parameters that allowed us to select clonal lines of A. marmorata, an endangered species. The first feature for our selection of clonal lines was to avoid masking the natural seed germination vigor using chemical or mechanical treatments of scarification or nutrient use in the phytagel agar that could influence germination. According to germination results, both lots of seeds kept in the laboratory for 12 and 18 months presented germination percentages of around 90% in less than ten days  medium. As a response to BAP, we found that 50% of the lines were individualized. Since this type of material is unsuitable for producing shoots, it was immediately discarded.Seventy seedlings (lines) derived from seeds were analyzed for micropropagation in MS+BAP , IBA (Indole Butyric Acid), BA (6-benzyl adenine), BAP (6-benzyl aminopurine), NAA , IAA (Indole-3-acetic acid), TDZ (Thidiazuron), Kinetin and Topolin. Among these, 2,4D-BA or 2,4D-IBA are the most commonly utilized [Several micropropagation protocols have been designed for utilized ,49,50,51−1 or 22.2 µM produced the highest number of shoots (83.4 ± 4.56) in A. marmorata clonal line AM32 and the fewest in line AM24 (6.6 ± 2.07), indicating a heterogeneous response between clonal lines to BAP cytokinin  with a tendency to be easily individualized. However, for plant production purposes, rooting during shoot multiplication attenuates shoot multiplication and/or upregulates cellular processes to sustain plant growth, consequently increasing production costs.In this work, we compared the shoot multiplication of 10 selected lines using the direct organogenesis method, which involves applying only BAP cytokinin as a shoot inducer in a vitamin-free MS medium for eight weeks. We found that BAP at 5 mg Lytokinin . Our finA. salmiana, through a selection of seedlings 60 d after seed germination formed 3 and 3.5 shoots with BAP concentrations of 0.5 and 10 mg L−1, which correspond to the minimum and maximum BAP concentration in MS + vitamins. The shoot induction increases when BAP (10 mg L−1) was combined with 0.04 mg L−1 of 2,4D-BA, yielding 14 ± 0.70 shoots. A significant number of shoot buds (32.8 ± 4.2) was reported by Rios-Ramirez et al. [A. angustifolia and a BAP and IAA combination (4:1 mg L−1). Moreover, Dominguez et al. [A. cupreata (10.5) at a BAP concentration of 1.5 mg L−1. For shoot induction in agave species A. difformis, A. karwinskii, A. obscura and A. potatorum, the optimal BAP concentration was 1.5 mg L−1, producing shoots ranging from 4.7 (A. obscura) to 6.4 (A. difformis). Furthermore, 13.32 µM BAP in MS and MS modified with half nutrient formulation induced 18.5 ± 1.9 and 13.4 ± 0.5 shoots in A. americana seedlings after 50 days [For in vitro propagation of Agave, the BAP cytokinin alone or combined with other substances has produced different efficiencies for shoot induction. For example, Puente-Garza et al.  found thz et al.  using stoot buds .8 ± 4.2  50 days .A. marmorata clonal lines to the same BAP concentration. For example, AM24 exhibited attenuated shooting and rapid induction of tissue senescence, while M14d-e displayed reduced shoot numbers without senescence.BAP is an aromatic cytokinin with good shoot induction because it is quickly metabolized in plant tissues. However, it has side effects at high concentrations, such as decreasing the shoot number or length and inducing apoptosis in plant tissues and cell cultures ,54,55. IRecently, there has been an emerging interest in studying plant responses to cytokinins. These hormones are perceived via a phosphorelay similar to the two-component systems bacteria use to sense and respond to environmental stimuli. In plants, cytokinin responses involve two-component perception systems  responding to biotic and abiotic stresses ,58. TherA. marmorata mass propagation and conservation.Our results reveal differences in shoot induction and multiplication among the clonal lines, even under identical in vitro culture conditions. From the plant production perspective, the three selected clonal lines (AM31. AM32 and AM33) tolerated a nutrient-free environment such as vermiculite and beach sand. Therefore, the behavior of these lines could contribute to A. marmorata, we quantified the content of flavonoids, phenolic compounds and saponins. These phytochemicals are high-value co-products in agaves [Agave species, from plants grown in the field or via in vitro micropropagation [A. marmorata “hijuelo” maintained in pots for three years compared to the clonal lines  at different wavelengths, differences in the compounds in the extracts were identified (We employed chromatographic methods to detect differences in the concentrations and types of secondary metabolites accumulated in the shoots of the three entified . Using Mentified . Flavonom/z) support the presence of these molecules. Those compounds were previously reported by Puente-Garza et al. [We also identified phenolic compounds such as caffeic acid, catechin, gallic acid and sinapic acid. Furthermore, three saponins, possibly tigogenin and hecogenin , were tea et al. ,72.A. marmorata is a phenotypic characteristic that distinguishes it from other Agave species and is present in an early stage of plant age.Finally, anthocyanins, including Cyanidin 3,5 Diglucoside, Delphinidin 3,5-O-Diglucoside and Delphinidin-3-O-Glucoside, stood out as compounds with the most significant differences between lines. These molecules have been previously reported in several species of wild agaves and attributed to plant protective functions. The presence of anthocyanins in A. marmorata shoots. Indeed, previous studies reported the usefulness of MALDI-TOF-MS in profiling phenolic compounds in plants [m/z 250–800 Da) [m/z between 484–656 Da were associated with glycosylated anthocyanins, and those with m/z 295, 303, 325, 482, 484, 496 and 595 corresponded to glycoside flavonoids with hexoses and pentoses such as Quercetin, Myricetin and Kaempferol [m/z > 1000 Da corresponded to saponins [Typically, MALDI-TOF mass spectrometry is used to study large biomolecules , and ESI-MS is used to analyze the chemical profile of low molecular weight molecules (<1000 Da). However, this paper demonstrates that operating the MALDI-TOF-MS in the reflectron mode facilitates the assessment of flavonoids, phenolic compounds, and anthocyanins in n plants ,74,75 an–800 Da) . Ionizatempferol . Additiosaponins .A. tequilana growth under limited nitrogen conditions [Endophyte microorganisms (bacteria and fungi) have been recognized for their role in plant growth and protection against abiotic and biotic stress ,79. Plannditions .E. cloacae and the clonal lines and specificity of A. xylosoxidans and P. aeruginosa for A. marmorata. According to Sneck et al. [Our findings indicate incompatibility between k et al. , one of k et al. .E. cloacae is an endophyte widely distributed among plants and their seeds [E. cloacae SEA02 induced a noticeable biomass accumulation in micropropagated A. tequilana plants, providing organic nitrogen once the bacterium colonized the root (Garcia-Ochoa manuscript in preparation). Additionally, in micropropagated banana plants, highly colonizing endophytic E. cloacae (C2 strain) inoculation enhanced plant growth, transferring nutrients from the soil to the roots, thus, making it an essential bacterium for bananas [The ir seeds ,86,87,88 bananas .A. tequilana with E. cloacae SEA02 rapidly induces H2O2 production in the whole plant in just a few minutes [Previously, it was demonstrated that inoculating the roots of  minutes . This fa minutes ,86.B. tequilensis is an endophytic bacterium contained in the seeds of A. tequilana. It greatly affects the AM33 line, especially root formation (number and length). In the other clonal lines, its effects were quite limited. It has been demonstrated that when grown under limited N and P, B. tequilensis provides organic N to A. tequilana plants [B. tequilensis has been shown to transfer organic N by isotopically marked proteins and nucleic acids of bacteria using 15NH4Cl as the nitrogen source. After several inoculations, the nitrogenous material of A. tequilana cells began to incorporate 15N, even in the chlorophyll, where the molecule’s four nitrogen atoms are isotopically labeled [a plants . B. tequ labeled . Herein,E. cloacae resulted in more dead plants; thus, it is difficult to ascertain whether E. cloacae behaves like a pathogen or does not support the growth of plants since no signs of bacterial rot or disease were observed and no significant differences in death among the three clonal lines. According to Garcia-Ochoa from our research group (manuscript in preparation), the E. cloacae SEA02 strain has plant-promoting properties such as nitrogen fixation and ACC deaminase activity. However, this endophyte does not produce auxins, likely accounting for the lack of root stimulation in A. marmorata, which hindered several growth parameters. In contrast, Bt, Ax and Ps strains produce IAA-type auxins.The effects of bacterial inoculations on the plant growth of each clonal line are summarized in We did not evaluate if genetic differences between the selected clonal lines, but we did observe some differences in the plants’ growth responses due to the endophyte inoculation. The highest response was observed on AM32, especially concerning root formation and growth. Root growth induction is essential for agaves because it provides anchorage in the soil and water and nutrient absorption, fundamental processes in eroded and dried soils.A. xylosoxidans strain contributed the greatest to root formation in all clonally propagated lines, producing 16 new roots. A study by Aguilar-Jimenez and Rodriguez [A. marmorata seedlings grown in an MS medium supplemented with 10 mg/L of the Indole Acetic Acid (IAA) auxin. The A. xylosoxidans N-strain used in this work is a diazotrophic endophyte, and a phosphate solubilizer previously isolated from A. marmorata seedlings developed 10 d after seed germination [The odriguez  reportedmination .A. xylosoxidans as an endophyte and plant growth-promoting bacteria (PGPB). Benson et al. [A. xylosoxidans strain AUM54 combined with indole-3-butyric acid (IBA) promoted shoot growth, root length, the number of roots and stress tolerance compared to control plants under micropropagation conditions of the endangered medicinal plant Naravelia zeylanica. The authors also showed that A. xylosoxidans plays a role in increasing plant survival during greenhouse acclimatization. Another study demonstrated the biological control effect against the fungus Magnaporthe grisea exerted by the same strain of A. xylosoxidans when inoculated in rice seeds. In addition to decreasing the colonization of the phytopathogenic fungus, there was an increase in the germination rate, seedling vigor index and yield of inoculated rice plants without disease conditions [Few published studies have demonstrated the potential of n et al.  reportednditions .A. xylosoxidans SQU-1, rhizobacteria strain isolated from date palms (Phoenix dactylifera L.) grown under salinity, enhanced date palm seed germination and Arabidopsis growth under normal and saline conditions. The dry weight gain in AM32 line plants is possibly related to the contribution of Ax to nutrient supply. However, the mechanisms and responses of the clonal line to biotization with A. xylosoxidans still need to be explored.Furthermore, Jana and Yaish  recentlyPlant micropropagation often results in differences in the endophyte microbiome compared to plants grown conventionally. Indeed, many beneficial microorganisms are eliminated through the micropropagation protocols used. Biotization with endophytes is an attractive new strategy to protect plants that are micropropagated, avoiding mortality when transferred from in vitro to ex vitro conditions. Micropropagated plants inoculated with endophyte bacteria exhibit physiological and developmental changes and pathogen resistance ,94,95,96A. marmorata seeds. The application of this process could contribute to improving Agave reintroduction into the forest for conservation and ecosystem restoration purposes. We selected the clonal lines based on seed germination capacity, seedling stability, and efficient shoot multiplication in the presence of the BAP cytokinin. The variations in multiplication capacity and stable tissue formation observed in different clonal lines may indicate genetic variability in seed populations. These genetic variations could be exploited for the production of high-quality plants from seeds with different capacities, such as fast propagation, higher adaptation to stress, and growth under nutrient limitation, but allows for maintaining different genetics in the plantations to satisfy the demand for plants without putting a natural resource at risk.A micropropagation protocol was developed using axillary bud proliferation to multiply clonal lines derived from A. xylosoxidans, an endophytic bacteria of A. marmorata.We sought to find other differential features between lines through microbiological approaches and phytochemical analysis. For example, the AM32 line presents a differential response to inoculation with seed-vectored endophytic bacteria, and AM31 differentially accumulates phenolics. The responses to inoculation highlighted the compatibility of A. marmorata shoot extracts. This observation opens the possibility of using micropropagation of A. marmorata as a source of bioactive compounds, consequently promoting the conservation of this endangered Agave species. To our knowledge, this is the first report of secondary metabolites extracted from the vegetal material of A. marmorata. Further studies need to be undertaken to determine if these metabolites can be used as biomarkers of plant stage and/or the response to stresses caused by cytokinins or environmental factors.Furthermore, compatibility and incompatibility responses to inoculation in three of the ten clonal lines indicated plant-bacteria specialization, which could have incompatibility consequences even in plants of the same species but with slight genetic differences. We also identified some phytochemicals considered bioactive compounds accumulated in the


Pteris vittata L. and Fallopia japonica, two metal-tolerant plants from northern Vietnam, on six antibiotic-resistant Stenotrophomonas maltophilia strains possessing efflux pump resistance mechanisms that were isolated from soil and clinical samples. The chemical composition of aqueous and dichloromethane (DCM) fractions extracted from P. vittata and F. japonica was determined using UHPLC-DAD-ESI/QTOF analysis. The antibacterial and efflux pump inhibitory activities of the four fractions were evaluated for the six strains  using a microdilution assay at fraction concentrations of 62.5, 125, and 250 μg/mL. The DCM fraction of F. japonica exhibited remarkable antibacterial activity against strain 0366, with a MIC of 31.25 μg/mL. Furthermore, this fraction also significantly decreased gentamicin MIC: four-fold and eight-fold reductions for BurA1 and BurE1 strains, respectively (when tested at 250 μg/mL), and two-fold and eight-fold reductions for K279a and BurE1 strains, respectively (when tested at 125 μg/mL). Pure emodin, the main component identified in the DCM fraction of F. japonica, and sennidine A&B only reduced by half the MIC of gentamicin (when tested at 30 μg/mL). Our results suggest that the DCM fraction components of F. japonica underground parts may be potential candidates for new bacterial efflux pump inhibitors (EPIs).During the process of adapting to metal contamination, plants produce secondary metabolites that have the potential to modulate multidrug-resistant (MDR) phenotypes; this is achieved by inhibiting the activity of efflux pumps to reduce the minimum inhibitory concentrations (MICs) of antimicrobial substrates. Our study evaluated the effect of secondary metabolites of belowground parts of  Stenotrophomonas maltophilia is a Gram-negative bacterium found in the environment and considered an opportunistic pathogen. Its various strains are found both in natural and anthropogenic environments, such as soil contaminated with heavy metals, causing numerous infections in hospitals [S. maltophilia strains possess a high capacity for resistance against different antibiotics, including aminoglycosides, beta-lactam, carbapenem, quinolones, and polymixines [S. maltophilia are multidrug-resistant (MDR), and phenotypically and taxonomically homogeneous [ospitals . Clinicaymixines . Moreoveogeneous . S. maltophilia has developed multiple resistance mechanisms resulting in its intrinsically low sensitivity to antibiotics, to which multidrug efflux pumps contribute significantly causing its MDR phenotype [henotype . These phenotype . The ovehenotype ,3. Therehenotype . Furtherhenotype ,6. Fallopia japonica (Houtt.) Ronse Decr , also called Japanese knotweed, is known as a plant able to grow in metal-polluted soil. The proportion of dihydroanthracenone derivates found in F. japonica root extract, including torosachrysone, anthraquinone, and dianthrone, are increased by metal contamination [Stenotrophomonas and the concentration of emodin, a bioactive compound identified in root extract of F. japonica. This compound protects plants against toxic factors but also possess an antimicrobial effect. The research showed that the combined impact of emodin could assist F. japonica growth under the dominance of Stenotrophomonas in the root microbiome [mination . Recent crobiome .Pteris vittata L., commonly found in tropical and subtropical areas such as Southeast Asia, Africa, and Australia, has been reported to be an excellent metal hyperaccumulator. In 2001, Ma et al. proposed that P. vittata in Florida could hyperaccumulate up to 22,630 mg/kg in leaves [P. vittata could grow and develop normally on Zn-contaminated soil with the ability to accumulate Zn up to 737 mg/kg in the leaves [P. vittata and some other ferns were employed; the concentrations of As that accumulated in the roots and leaves reached 6042 ppm and 3756 ppm. As a result, they could tolerate soils highly contaminated with 15,146 ppm As, 5000 ppm Pb, and 1200 ppm Cd [P. vittata dominating Thai Nguyen mining sites has been revealed using a combination of ionomics and metabolomics analysis [P. vittata possesses antioxidant, hypoglycemic, anti-inflammatory, antiplatelet aggregation, and antitumor activities [P. vittata metabolome [Burkholderia spp., Acinetobacter spp., or Cupriavidus spp. in the rhizosphere of P. vittata [n leaves . Moreovee leaves . Therefoe leaves ,10,11,12analysis . In the tivities . Recentltabolome , the subtabolome , and thetabolome . The antP. vittata and F. japonica on six S. maltophilia strains isolated from soils and clinical samples. The present study evaluates the antibacterial effect and the efflux pump inhibitory activities of secondary metabolites extracted from the aqueous and dichloromethane (DCM) fractions of belowground parts of P. vittata and F. japonica extracts against six strains of S. maltophilia that potentially expressed efflux pumps were determined. In this study, the chemical composition, minimum inhibition concentration (MIC), and efflux pump inhibitory activities of P.vittata and F. japonica were A-type polymeric procyanidins together with caffeoyl-quinic acid derivatives, and trans-reseveratrol derivatives together with B-type polymeric procyanidins, respectively. In the P. vittata DCM fraction, an unidentified diterpene was the only compound tentatively annotated, whereas in the F. japonica DCM fraction, emodin, emodin dianthrone isomers, physcion, torosachrysone, and fallopion isomers were the major secondary metabolites detected. The chemical composition of the four fractions is shown in The main components of the aqueous fractions of F. japonica which exhibited noteworthy antibacterial activity against strain 0366 with a MIC of 31.25 μg/mL  at 25 μg/mL, a commercial EPI. For strains K279a, BurA1, and BurE1 a reduction of gentamycin MIC was observed (with up to four-fold reduction for BurE1), whereas, for the three other strains , PAβN did not reduce gentamicin MIC . Previous studies reported high antibacterial efficacy of anthraquinones against Gram-positive bacteria such as Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus ATCC25922 or Salmonella anatum [Haemophilus parasuis of emodin extracted from F. japonica [F. japonica showed a moderate antibacterial effect when tested with S. maltophilia. This is not surprising considering that S. maltophilia strains are known to exhibit elevated multidrug resistance by forming a natural protective layer against the host immune defense system and several antimicrobial agents using intrinsic or acquired resistance mechanisms [S. maltophilia 0366, our unpublished data showed that this clinical strain was resistant to antibiotics of the β-lactam  and quinolone groups (ciprofloxacin and pefloxacin), but it was sensitive to a combination of a β-lactam and a β-lactamase inhibitor (ticarcillin + clavulanic acid), aminoglycosides group , polymyxin (colistin) and trimethoprim/sulfamethoxazole. In our study, three S. maltophilia strains  previously showed in vitro resistance to almost all the antibiotic classes including aminoglycosides, penicillin, carbapenems, polymyxin, and monobactam [S. maltophilia 0366 and other S. maltophilia strains may be one of the reasons justifying the low MIC (31.25 μg/mL) obtained for F. japonica DCM fraction against this strain. We suggest that the F. japonica DCM fraction could possess several secondary metabolites including anthraquinones and resveratrol derivatives that might be active against S. maltophilia 0366. However, the reasons for the sensitivity of this strain remain to be elucidated. Therefore, it is envisaged to isolate the compounds from the F. japonica DCM fraction and evaluate their antibacterial activity against the S. maltophilia 0366 strain in our future works. effects ,22,23. Wa anatum . Anotherchanisms ,27. For P. aeruginosa, K. pneumoniae, C. jejuni, E. coli, S. typhimurium, and E. aerogenes) and antibiotic  ranges [S. maltophilia strains , therefore the efflux pump inhibitory activity assay was not performed on these strains. They may employ different mechanisms of resistance than efflux pumps, as reported elsewhere [F. japonica with emodin, emodin dianthrone, physcion, and fallopion as its main components was the most active fraction tested. At the maximum concentration tested (250 µg/mL), this extract reduced gentamicin MIC on both clinical and environmental strains  (50=9.42 μM) [hefA, a gene encoding a member of the active efflux system in Helicobater pylori [Stenotrophomonas in F. japonica’s root microbiome either directly, by promoting the growth of Stenotrophomonas, or indirectly, by inhibiting the growth of other microorganisms to assist F. japonica growth [F. japonica were found to be raised under metal stress [Mycobacterium smegmatis [The importance of efflux pumps and their role in antibiotic resistance is increasing. As a result, the inhibition of multidrug efflux pumps is a promising approach to restore the efficacy of existing antibiotics as adjunctive therapies and thus contribute to the reduction of the emergence of MDR bacteria . In this) ranges ,31,32. Ir BurE1) . Emodin 9.42 μM) , and inh9.42 μM) . Specifir pylori . A recenmegmatis .P. vittata L did not significantly reduce gentamicin’s MIC. However, we were able to identify diterpenes: a group of terpenoids from the DCM fraction of P. vittata. Several terpenoids were reported to exhibit a broad spectrum of antibacterial activity [P. vittata is commonly used in the treatment of dysentery and diarrhea caused by bacterial infections [P. vittata extract inhibited E. coli, B. subtilis, P. aeruginosa, S. aureus, and Proteus vulgaris at different concentration levels [P. vittata fractions did not show significant antibacterial and efflux pump inhibitory activity. Therefore, optimizing the P. vittata extraction process will be necessary for future research. Regarding efflux pump inhibitory activity, the DCM fraction of activity . In tradfections ,36. In dn levels . In our S. maltophilia, commonly found in the environment, is an opportunistic bacterial pathogen that is resistant to common antibiotics and causes several infections in hospitals [S. maltophilia strain with high MIC to several antibiotics was found to coordinately hyper-express the Resistance-Nodulation-Division (RND) family efflux pumps SmeZ and SmeJK [S. maltophilia, is the result of the flavonoid binding to the transcriptional repressor SmeT [S. maltophilia strains, including K279a, BurA1, and BurE1 which have previously shown in vitro resistance to almost all the antibiotic classes including aminoglycosides, penicillins, carbapenems, polymyxin and monobactam [-TolC pumps played an important role in the extrusion of aminoglycosides in S. maltophilia [macA and macB were identified in all three strains [S. maltophilia to aminoglycosides [MacABCsm, SmeIJK, SmeYZ, and SmeOP-TolC in the S. maltophilia BurA1, BurE1, and K279a strains using quantitative real-time reverse transcription PCR. It is also important to compare them to the expression levels of three other tested S. maltophilia strains  and the negative control  to confirm the critical role of these efflux pump genes in antibiotic resistance contributing to MDR phenotypes in these strains. Most importantly, the main components of the DCM fraction will be purified to evaluate their individual antibacterial and efflux pump inhibitory activities.ospitals ,38. The ospitals ,38. The ospitals ,39,40. And SmeJK ,42. MDR sor SmeT . Additiotophilia ,45,46. Fycosides . TherefoPlant materialsP. vittata L. and F. japonica belowground parts were collected in Dai Tu, Thai Nguyen, and were identified by Dr. Do Van Hai at the Institute of Biological Resources, Vietnam Academy of Science and Technology. The voucher of specimens was deposited in the Laboratory of Life Sciences Department, Hanoi University of Science and Technology, Vietnam Academy of Science and Technology.Bacterial and growth conditionsStenotrophomonas maltophilia strains . Among them, 502, 0366, and K279a were obtained from clinical samples [The tested bacteria were various  samples  while th samples . These s8 CFU/mL) in sodium chloride 0.9% of each strain was obtained using a densimeter . These suspensions were then diluted in nutrient broth to obtain stock solutions containing 106 CFU/mL.Bacterial strains were grown in nutrient agar medium , and incubated at 28 °C for 24 h. A concentration of 0.8 McFarland (10Preparation of plant extractsP. vittata L. and F. japonica were grinded after freeze-drying and extracted three times with solvent mixture of MeOH and distilled water 1:1 (v/v), then with 100% MeOH . The two solvent extracts were combined and the MeOH was evaporated under reduced pressure . The extracts were then transferred into a separatory funnel and partitioned against equal volumes of DCM to afford two fractions: the aqueous fraction and the DCM-soluble fraction. All extracts were concentrated until a constant weight was obtained.The dried belowground parts of Chemical composition of plant fractionsPteris spp., Fallopia spp., or species in other genera.The main components of the aqueous and DCM phases for the two tested plants were analyzed using UHPLC-DAD-ESI/QTOF analysis performed on an Agilent Infinity® 1290 system with a UV/vis DAD G4212A detector and an q-TOF 6530 detector controlled using the MassHunter® software . The putative annotation of the main compounds in each fraction was based on UV, HRMS, and MS-MS spectra, and relative retention times. Data were compared with previous literature reports for compounds in Minimum inhibitory concentration (MIC) determinationP. vittata L. and F. japonica dried roots were evaluated by means of the micro-dilution assay against the six bacterial strains of S. maltophilia  [6 colony forming units (CFU)/mL in nutrient broth were added to the wells to obtain final concentrations of each fraction between 15.625 and 2000 μg/mL. The plates were sealed and incubated at 28 °C for 24 h. Bacterial growth was assessed by determining the OD of the solution in each well at 600 nm. The minimum inhibitory concentration (MIC) was determined as the lowest concentration at which the bacterial growth was inhibited by over 90%. Triplicate wells were measured and the data were then averaged. In cases where complete inhibition occurred at the lowest concentration (15.625 μg/mL), further serial dilutions were performed until a MIC endpoint was reached. Sulfamethoxazol  (at concentrations of 20 and 40 mg/mL) was used as the positive antibacterial control. The final DMSO concentration in each well was 0.5% per well, and sterile broth was used as negative controls. At this percentage, DMSO had no effect on bacterial growth.The antibacterial activities of both aqueous and DCM fractions extracted from d BurE1) ,40. The Efflux pump inhibitory activity evaluation6 CFU/mL. The microtiter plates were incubated at 28 °C for 24 h before the results were recorded. Bacterial growth was determined by measuring the OD of the solution in each well by using a TECAN Infinite® 200 Pro microplate reader . MIC changes of four-fold or greater were considered significant. The combination of gentamicin and phenylalanine-arginine β-naphthylamide (PAβN)  at a concentration of 25 μg/mL was used as a positive control. Ultrapure water and a sterile NB medium were used as negative controls and had no effect on bacterial growth. The tested extracts could be considered as EPIs if they reduced the gentamicin MIC by at least four-fold.The MIC of the tested antibiotic was determined by two-fold serial broth microdilution in 96-well plates in the presence or absence of each extract at the indicated concentration, which was four-fold lower than its MIC as determined in the above-mentioned test. Gentamicin  was the antimicrobial agent used at concentrations that varied from 0.625 to 80 μg/mL. Each well contained 5 μL of the antibiotic, 5 μL of the extract, 90 μL of the NB medium, and 100 μL of the NB inoculum containing 10S. maltophilia strains of aqueous and DCM fractions extracted from P. vittata L. and F. japonica plants native to the northern Vietnam. The F. japonica DCM fraction, with emodin, emodin dianthrone, physcion, torosachrysone, and fallopion isomers as major components, exhibited the most significant activity, reducing the MIC of gentamicin four-fold for the BurA1 strain, eight-fold for the BurE1 strain at 250 μg/mL, and four-fold for the BurE1 strain at 125 μg/mL. The separation, purification, and identification of potential herbal-derived compounds with efflux pump inhibitory activities will undoubtedly foster a better understanding of the application of EPIs when combined with antibiotics for the treatment of (multi)drug-resistant bacterial infections.Our new data provide significant insights into the chemical composition and antibacterial efficacy (MIC and efflux pump inhibitory activity) of six


Pulses are a group of leguminous crops that are harvested solely for their dry seeds. As the demand for plant-based proteins grows, pulses are becoming important food crops worldwide. In addition to being a rich source of nutrients, pulses also contain saponins that are traditionally considered anti-nutrients, and impart bitterness and astringency. Saponins are plant secondary metabolites with great structural and functional diversity. Given their diverse functional properties and biological activities, both undesirable and beneficial, saponins have received growing attention. It can be expected that redirecting metabolic fluxes to control the saponin levels and produce desired saponins would be an effective approach to improve the nutritional and sensory quality of the pulses. However, little effort has been made toward understanding saponin biosynthesis in pulses, and, thus there exist sizable knowledge gaps regarding its pathway and regulatory network. In this paper, we summarize the research progress made on saponin biosynthesis in pulses. Additionally, phylogenetic relationships of putative biosynthetic enzymes among multiple pulse species provide a glimpse of the evolutionary routes and functional diversification of saponin biosynthetic enzymes. The review will help us to advance our understanding of saponin biosynthesis and aid in the development of molecular and biotechnological tools for the systematic optimization of metabolic fluxes, in order to produce the desired saponins in pulses. Pulses are leguminous crops that are harvested strictly for their dry seeds, including peas, dry beans, lentils, chickpeas, faba beans, cow peas and pigeon peas. They are consumed worldwide, and have received growing attention due to both nutritional and environmental benefits. Pulses provide a rich source of protein, fiber, minerals and vitamins, as well as a healthy diet of low glycemic index that is also cholesterol-free and gluten-free. Pulses also contain an array of phytochemicals with various potential benefits for human health . SaponinSaponins are a structurally diverse group of specialized plant terpenoids in many plant species, consisting of either steroidal or terpenoidal aglycones linked to oligosaccharide moieties ,4. In diIt is generally believed that saponins contribute to the bitter and astringent taste of pulse-based food products . UnderstAphanomyces euteiches, or Rhizoctonia solani and Fusarium oxysporum) significantly increased the saponin content in grains of two pea (Pisum sativum L.) cultivars, Crécerelle and Firenza [Saponins display a wide range of biological activities such as anti-microbial or anti-insect repellent activity, being produced as plant defense molecules in response to biotic threats, pathogens and herbivores . Being a Firenza . Additio Firenza . Given t Firenza . In addi Firenza ,13,14,15In the context of human health, saponins exhibit a wide spectrum of pharmacological properties, including anti-cholesterolemic, anti-cancer and hemolytic activities ,4,9,16. Glycine max), peas (Pisum sativum) and chickpeas (Cicer arietinum) [Lens culinaris Medik.), chickpea (Cicer arietinum L.), faba bean (Vicia faba) and dry bean (Phaseolus vulgaris L.) [Soyasaponins, belonging to the oleanane triterpenoid group, are composed of soyasapogenol and oligosaccharide moieties, and are abundantly present in in the seeds of legumes such as soybean and pulses ,9. Four ietinum) . The conietinum) . Likewisietinum) . Howeverietinum) . One posaris L.) . Both graris L.) . Seed siaris L.) .Processing and cooking reduce the total saponins, although to various degrees, in different pulse grains ,31 and aIn this review, we will focus on the biosynthesis of triterpenoid saponins, since they are the class present in the pulses. Saponin biosynthesis involves sequential actions of three key classes of enzymes encoded by multi-gene families . The bioMedicago truncatula and in soybeans, only a few genes have been cloned and characterized in pulse crops (Although significant progress has been made on saponin biosynthesis in the model legume se crops . Many geP. sativum) OSC sequences reported by Vernoud et al. [M. truncatula, soybean (G. max) and pulse crops, including the common bean (Phaseolus vulgaris L.), tepary bean (Phaseolus acutifolis A. Gray), lima bean (Phaseolus lunatus), cowpea , lentil (Lens culinaris Medik.) and chickpea (C. arietinum) , cycloartenol synthase (CAS), lanosterol synthase (LAS), lupeol synthase (LUP) and several mixed-function OSCs that produce diverse products. These OSCs have specialized functions and make different major products . A neighd et al. , togetheietinum) . These Ouncatula  and Barbvulgaris , suggestvulgaris .bas1, in two different genetic backgrounds led to the identification of the PsBAS1 gene (Psat7g264880) encoding a β-amyrin synthase that was expressed in several tissues, including the developing seeds [PsBAS1 was much higher in the seed embryo than the seed coat, which coincided with the accumulation patterns of both DDMP and type B saponins. The expression of PsBAS1 was first detected at 19 days after pollination (dap) and increased as the seed matured with the peak at 29 dap [PsBAS1 throughout the pea seed development was in agreement with the accumulation pattern of saponin content, which was strongly increased in the late stage, indicating a key role of PsBAS1 in the biosynthesis of saponin [P. sativum, with one encoding β-amyrin synthase (PSY) [Psat7g264880, and the other encoding mixed amyrin synthase (PSX) to produce both α- and β-amyrin [Psat4g188800 at the amino acid level. A cDNA encoding cycloartenol synthase was isolated from pea seedlings [Psat1g051480. To our knowledge, OSCs have not been characterized in other pulse crops. Functional characterization of these OSCs is necessary to advance our knowledge of saponin biosynthesis and to facilitate the development of molecular tools to modify saponin content in pulse crops.A few OSCs have been isolated and functionally characterized in peas. Characterization of two ethyl methanesulfonate (EMS)-induced pea mutants, ng seeds . The expt 29 dap . The gen saponin . Two trise (PSY)  that wasβ-amyrin , which heedlings , with 99Leguminosae family, and have yet to be found outside the Leguminosae. Unlike CYP93E, the CYP72A subfamily is distributed across the entire plant kingdom [The second diversifying step for triterpenoid biosynthesis occurs when P450s catalyze the site-specific oxidation of the cyclized skeleton, primarily through the introduction of hydroxyl, carboxyl or epoxy groups . A largeP. sativum, M. truncatula, L. culinaris Medik. and C. arietinum L. are clustered together, suggesting that these species likely share a common ancestry. The CYP93E subfamily catalyzes the C-24 oxidation of the triterpene backbone (β-Amyrin) to produce 24-OH-β-amyrin during the biosynthesis of triterpenoid saponins [M. truncatula) and CYP93E8 (P. sativum) were demonstrated in a heterologous host (Saccharomyces cerevisiae) [M. truncatula ortholog CYP72A61v2 transform 24-OH-β-amyrin into the major soyasaponin aglycone, soyasapogenol B, by catalyzing the hydroxylation at the C-22 position [G. max) [Glyma15g39090 encoding the cytochrome P450 enzyme, CYP72A69, led to metabolic switching from undesirable group A saponins to beneficial DDMP saponins in soybean [Phylogenetic analysis of putative P450s involved in soyasaponin biosynthesis distinguishes three distinctive clades . Only a saponins . Differeevisiae) . The CYPposition . Soyasap[G. max) ,52,53. S soybean . The ideM. truncatula) can glycosylate certain isoflavones and the flavonol quercetin, in addition to triterpenoids [M. truncatula, UGT73K1 was demonstrated to have specificity for hederagenin and soyasapogenols B and E, while UGT71G1 has specificity for medicagenic acid [Glycosylation is the last modification step in the biosynthesis of saponins. This sugar conjugation step is important for the biological activities and chemical properties of saponins by regulating triterpenoid aglycones, including increasing their stability, water solubility and cell compartmentalization . The glyrpenoids . Most ofrpenoids . In M. tnic acid . In soybnic acid . The varnic acid .P. sativum, M. truncatula, L. culinaris Medik. and C. arietinum L. are clustered together. Multiple members are present in each of the subfamilies. However, further experimental studies are required to confirm whether they are involved in saponin biosynthesis, as well as what their functional mechanism is. It is not completely clear which enzymes are involved in the biosynthesis of DDMP soyasaponins. However, a member of UGT73B4, encoded by the gene Glyma.16G033700 in soybeans, has been suggested to function as a DDMP transferase, catalyzing the production of DDMP saponins from group B saponins [A neighbor-joining phylogenetic tree for the subfamilies of putative UGT71, UGT73 and UGT91 orthologs in pulse crops  shows thsaponins  and/or asaponins .Panax quinquefolius L) [M. truncatula, two JA-inducible bHLH TFs, TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (TSAR1) and TSAR2 positively regulate non-hemolytic and hemolytic triterpenoid saponin biosynthesis, respectively [Medtr2g104650, regulates the expression of CYP88A13 in the developing seeds of M. truncatula, a cytochrome P450 that catalyzes the final oxidation step of the hemolytic saponin biosynthesis branch [M. truncatula by suppressing the activities of TSAR1 and TSAR2 [The biosynthesis and accumulation of saponin are subjected to the transcriptional, developmental and spatiotemporal modulation . Prior solius L) . In the ectively . A seed-s branch . Althougs branch . Comparend TSAR2 .Gleditsia sinensis Lam. (Leguminosae) [In addition to TFs, microRNAs (miRNAs) have been reported to be involved in terpenoid biosynthesis and accumulation in plants . The miRminosae) . HoweverQuillaja brasiliensis when exposed to abiotic elicitors such as salicylic acid, jasmonic acid, UV light and mechanical damage [The dynamic accumulation of secondary metabolites, including saponins, in response to environmental stimuli is an adaptive strategy of the plants which allows them to survive under adverse environmental conditions. Abiotic environmental factors, such as temperature and precipitation, affect the accumulation patterns of saponins, including their content and composition . Increasl damage . Furtherl damage . With inTriterpenoid saponin biosynthesis in pulses is poorly understood, and many pathway genes remain uncharacterized. Rapid advances in next generation sequencing technology promote the increasing availability of genome and pan-genome sequences of multiple crop species, which no doubt accelerates the discovery of new genes/alleles involved in saponin biosynthesis. Integrated applications of the genome, transcriptome, proteome and metabolome are opening up unprecedented opportunities for functional genomics to elucidate the molecular mechanism underlying triterpenoid saponin metabolism in pulse crops. In conjunction with exploiting natural gene variants in diverse germplasms, genetic engineering with CRISPR and/or molecular breeding for targeted production of saponins will facilitate the development of the varieties with balanced benefits to plant health, nutritional quality for human and animal consumption, and palatability for pulse food products.


Anastomosis for gastrointestinal reconstruction has been contentious after low anterior resection of rectal cancer for the past 30 years. Despite the abundance of randomized controlled trials (RCTs) on colon J-pouch (CJP), straight colorectal anastomosis (SCA), transverse coloplast (TCP), and side-to-end anastomosis (SEA), most studies are small and lack reliable clinical evidence. We conducted a systematic review and network meta-analysis to evaluate the effects of the four anastomoses on postoperative complications, bowel function, and quality of life in rectal cancer.I2). The interventions were ranked according to the surface under the cumulative ranking curve (SUCRA) to compare each outcome indicator.We assessed the safety and efficacy of CJP, SCA, TCP, and SEA in adult patients with rectal cancer after surgery by searching the Cochrane Library, Embase, and PubMed databases to collect RCTs from the date of establishment to May 20, 2022. Anastomotic leakage and defecation frequency were the main outcome indicators. We pooled data through a random effects model in a Bayesian framework and assessed model inconsistency using the deviance information criterion (DIC) and node-splitting method and inter-study heterogeneity using the I-squared statistics (SEA = 0.982), followed by the CJP group (SUCRACJP = 0.628). The defecation frequency in the SEA group was comparable to those in the CJP and TCP groups at 3, 6, 12, and 24 months postoperatively. In comparison, the defecation frequency in the SCA group 12 months after surgery all ranked fourth. No statistically significant differences were found among the four anastomoses in terms of anastomotic stricture, reoperation, postoperative mortality within 30 days, fecal urgency, incomplete defecation, use of antidiarrheal medication, or quality of life.Of the 474 studies initially evaluated, 29 were eligible RCTs comprising 2631 patients. Among the four anastomoses, the SEA group had the lowest incidence of anastomotic leakage, ranking first (SUCRAThis study demonstrated that SEA had the lowest risk of complications, comparable bowel function, and quality of life compared to the CJP and TCP, but further research is required to determine its long-term consequences. Furthermore, we should be aware that SCA is associated with a high defecation frequency.The online version contains supplementary material available at 10.1186/s12957-023-02977-z. Colorectal cancer is the third most common cancer globally and the second largest cause of cancer fatalities, with rectal cancer accounting for at least one-third of all cancer fatalities . TransabConventional straight colorectal anastomosis is associated with numerous complications and is prone to severe bowel dysfunction –12. To iPrevious Cochrane systematic reviews and meta-analyses have concluded that the four anastomoses have similar complication rates. However, CJP is better than straight colorectal anastomosis (SCA) in bowel function, with no difference from SEA or TCP , 18. Thehttps://www.crd.york.ac.uk/prospero/), and we have reported the results according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) extended statement [The protocol for this study is registered with PROSPERO  the study participants were adults with rectal cancer who underwent surgical treatment, (2) at least two of the anastomosis techniques  were included in the study, (3) at least one of the primary outcome indicators (anastomotic leakage and defecation frequency) was included, and (4) the research type was English RCTs. Exclusion criteria were as follows: (1) non-randomized controlled trials, including reviews, retrospective studies, commentaries, and meta-analyses; (2) lack of available data or outcomes; and (3) duplicate publication of content. Two authors (MX and YZ) independently reviewed the entire text in accordance with the inclusion and exclusion criteria, consulted a third author (CWL) in case of disagreement, and decided on the inclusion of eligible studies at a conference.Two authors (MX and YZ) independently extracted the following data: (1) anastomotic leakage, (2) defecation frequency, (3) anastomotic stricture, (4) reoperation, (5) postoperative mortality within 30 days, (6) fecal urgency, (7) incomplete defecation, (8) use of antidiarrheal medication, and (9) quality of life. We recorded the results of bowel function outcomes at 3, 6, 12, and 24 months following stoma retraction (or without stoma surgery). We considered the most common and concerning anastomotic leakage and defecation frequency as the primary outcome indicators, and the rest were secondary outcome indicators. Anastomotic leakage is defined as a significant crack at the edge of the anastomosis, leakage of bowel contents seen in the pelvis on imaging or endoscopy, or purulent discharge from the pelvic drainage tube. The defecation frequency was determined based on the patient-described average number of daily bowel movements.Two authors (YBS and ZHP) independently assessed the risk of bias for all included studies using the revised Cochrane Rob2 tool, and studies with disagreements were resolved by a third author (CWL) . The folhttps://www.r-project.org/). Simultaneously, the meta package was used for pairwise analysis. Network meta-analysis results provided more accurate estimates and ranked various interventions to provide clinical recommendations compared to results from traditional pairwise analyses [P > 0.05, the direct comparison was considered to be in good agreement with the indirect comparison. We also evaluated the heterogeneity between studies using the I-squared statistics (I2) [I2 values was 0–100%, where 0–49% was low heterogeneity, 50–74% moderate heterogeneity, and 75–100% high heterogeneity. By calculating the surface under the cumulative ranking curve (SUCRA), we compared and ranked the safety and efficacy of various interventions. Higher ranking grades indicated lower perioperative complication rates or better bowel function. Due to the large variation in sample sizes of the included studies, sensitivity analyses on anastomotic leakage were performed to assess the reliability of the results, which included only studies with sample sizes greater than or equal to 20 in a single arm. To assess the publication bias of studies in the network meta-analysis, we used STATA 16.0  to generate a comparison-adjusted funnel plot and thus explore the impact of publication bias or other small-sample studies [We performed a network meta-analysis using the Bayesian framework employing gemtc and rjags packages in R4.2.0  , 27. The studies . In the We identified 471 articles by searching the database and three articles from the reference list for a total of 474 articles  and that for the CJP group was second (SUCRACJP = 0.628). These two anastomoses are the most secure. Additionally, 10 [SEA = 0.946 and 0.826), whereas the TCP group ranked fourth for both (SUCRASEA = 0.084 and 0.143).Twenty-four studies , 49–56 rally, 10 , 50, 54,ally, 10 , 55, andally, 10 –54, 56 rTCP = 0.727) and second (SUCRACJP = 0.720), respectively, followed by the SEA and SCA groups. However, there was no statistical difference among the groups , third (SUCRATCP = 0.556), and first (SUCRACJP = 0.722) positions, respectively. No intervention was found to be noticeably superior regarding fecal urgency, incomplete defecation, and use of antidiarrheal medication. The TCP group ranked first in fecal urgency and incomplete defecation , while the CJP group ranked second in both . The SEA group ranked first in the use of antidiarrheal medications (SUCRASEA = 0.718).Thirteen , 54, 57,SEA = 0.587), second (SUCRATCP = 0.659), and first (SUCRACJP = 0.743) positions, respectively. In addition, no substantial advantage was discovered for any specific intervention of fecal urgency, incomplete defecation, and use of antidiarrheal medication; nevertheless, the SEA group ranked best for both fecal urgency and use of antidiarrheal medication (SUCRASEA = 0.773 and 0.679).Twelve , 54, 57,SEA = 0.779) and second (SUCRACJP = 0.727), respectively. Similarly, there was no statistical difference in the use of antidiarrheal medication among the intervention groups, but the SEA and CJP groups were first (SUCRASEA = 0.776) and second (SUCRACJP = 0.708), respectively.Four , 34, 54 Nine studies assessed the quality of life of patients with different types of anastomoses. However, because the assessment scales and timing varied among the studies, we could not combine them for the analysis. We summarize all the results in the table below Table .Table 3SP = 0.02); no significant inconsistency was found in the other comparisons. Furthermore, we discovered low heterogeneity among studies on anastomotic leakage but generally high heterogeneity among studies on defecation frequency. We did not analyze it because of study limitations, but we speculated that it could be related to the variety of assessment scales and lack of objective evaluation.We only performed sensitivity analyses on data from anastomotic leakage because of the few studies. The DIC difference between models was reduced from 5.47 to 0.71 after removing studies with less than 20 sample sizes in a single arm, and the results were unchanged. The other outcome indicator models fit well, with the difference in DIC between the consistent and inconsistent models being less than 5. In our study, only the comparison of SCA with SEA in the fecal urgency outcome indicator at 12 months postoperatively was inconsistent . However, the study had a small sample size, short follow-up time, high missed follow-up rate, and variations in the proportion of radiotherapy among the groups, which somewhat diminished the credibility of the findings.Postoperative bowel function is an important indicator of clinical efficacy. According to this meta-analysis, there was no statistically significant difference in the defecation frequency among the groups at 3 and 24 months postoperatively, with the former possibly related to high rectal sensitivity and poor adaptation in the early postoperative period and the latter with adaptation having reached equilibrium over time. At 6 and 12 months postoperatively, there was no statistically significant difference in the defecation frequency among the CJP, TCP, and SEA groups. However, all were significantly lower than that of the SCA group, with the CJP group being first. The J-shaped pouch of the CJP and the lateral limb of the SEA, as well as coloplasty, improved fecal function to some extent after increasing the volume of the “new rectum” and decreasing peristaltic waves. In a prospective RCT by Okkabaz et al. , a 1-yeaRegarding fecal urgency, incomplete defecation, and the use of antidiarrheal medication, we did not find a consistent advantage for any of the anastomoses. Early studies found that CJP caused incomplete defecation, but after years of clinical practice, surgeons showed that a 5–6-cm J-shaped pouch has the same defecation frequency as an 8–10-cm J-shaped pouch and significantly reduces stool retention and the use of laxatives and enemas , 76. MosP = 0.007). The results of the available studies show that the four anastomoses have a similar impact on patient’s quality of life after surgery, with possible short-term quality of life benefits for patients with J-shaped pouches; however, CJP may result in poorer sexual function in male patients. Therefore, when selecting surgical procedures for patients, particularly male patients, clinicians should consider the patient’s specific situation and the surgeon’s preferences.One of the primary goals of evolving rectal cancer treatment is improving the patient’s quality of life. We discovered that severe postoperative complications and bowel dysfunction do not positively correlate with the quality of life. Most patients have a higher quality of life than expected from their bowel function outcomes. Table This systematic review integrated recent RCTs to rank postoperative complications and bowel function outcomes in rectal cancer by a network meta-analysis, giving an up-to-date basis for the anastomoses selection following rectal cancer surgery. However, some limitations limited the interpretation of our findings. First, the early trials included in this network meta-analysis were primarily single-center small-sample studies with short follow-up times and the inability to be double-blinded in clinical trials, which impacted the pooled results. Second, discrepancies in bowel dysfunction definition, measurement, and assessment scales resulted in a high level of inter-study heterogeneity in the comparison. Furthermore, some researchers have exaggerated the superiority of modified anastomosis, resulting in general publication bias in bowel function studies. Third, fecal incontinence, laxative use, enema use, anorectal pressure, overall survival, and disease-free survival, all of which are closely related to the quality of life of patients with rectal cancer, were not summarized in this network meta-analysis, and the conclusions may be limited.Future studies should first standardize the definition of various lesions. A comprehensive, universal, and well-validated international tool should be designed to facilitate communication and dissemination of research results between different regions. Second, more long-term multicenter RCTs are required to investigate the effects of different anastomoses on patient’s quality of life and sexual and urological function.Our systematic review of postoperative complications and bowel function in rectal cancer revealed that SEA has the lowest risk of complications and the best safety profile. Six months after surgery, the CJP and SEA were identical in defecation frequency, and other indicators of bowel function were similar. We also discovered that TCP is predisposed to complications, with SCA resulting in significantly more frequent postoperative bowel movements. Furthermore, the different anastomoses did not significantly impact the patient’s quality of life. However, the heterogeneity of bowel function studies undermines the credibility of the findings. In the future, more in-depth evaluations of the benefits and risks of various anastomoses in the long-term, high-quality studies are required.Additional file 1: Supplementary Table 1. Checklist of the PRISMA extension for network meta-analysis. Supplementary Table 2. Number of citations by each database searched. Supplementary Table 3. Characteristics of the 29 studies included in the network Meta-analysis. Supplementary Fig. 1. Risk-of-bias summary of the randomized controlled trials. Supplementary Table 4. Quality assessment of included randomized controlled trials. Supplementary Table 5. Results of global heterogeneity and local heterogeneity. Supplementary Table 6. Node-splitting analysis of inconsistency. Supplementary Table 7. Comparisons of the fitness of consistency and inconsistency models using deviance information criteria. Supplementary Fig. 2. Results of pairwise meta-analysis for postoperative complications. Supplementary Fig. 3. Results of pairwise meta-analysis for defecation frequency. Supplementary Fig. 4. Results of pairwise meta-analysis for bowel function. Supplementary Table 8A. Relative effects table for postoperative anastomotic leakage. Supplementary Table 8B. Rank probabilities for postoperative anastomotic leakage. Supplementary Fig. 5B. Comparison-adjusted funnel plot for postoperative anastomotic leakage. Supplementary Fig. 6A. Network plot for postoperative anastomotic stricture. Supplementary Table 9A. Relative effects table for postoperative anastomotic stricture. Supplementary Table 9B. Rank probabilities for postoperative anastomotic stricture. Supplementary Fig. 6B. Comparison-adjusted funnel plot for postoperative anastomotic stricture. Supplementary Fig. 7A. Network plot postoperative reoperation. Supplementary Table 10A. Relative effects table for postoperative reoperation. Supplementary Table 10B. Rank probabilities for postoperative reoperation. Supplementary Fig. 7B. Comparison-adjusted funnel plot for postoperative reoperation. Supplementary Fig. 8A. Network plot for postoperative mortality within 30 days. Supplementary Table 11A. Relative effects table for postoperative mortality within 30 days. Supplementary Table 11B. Rank probabilities for postoperative mortality within 30 days. Supplementary Fig. 8B. Comparison-adjusted funnel plot for postoperative mortality within 30 days. Supplementary Fig. 9A. Network plot for defecation frequency at 3 months postoperatively. Supplementary Table 12B. Rank probabilities for defecation frequency at 3 months postoperatively. Supplementary Fig. 9B. Comparison-adjusted funnel plot for defecation frequency at 3 months postoperatively. Supplementary Fig. 10A. Network plot for fecal urgency at 3 months postoperatively. Supplementary Table 13A. Relative effects table for fecal urgency at 3 months postoperatively. Supplementary Table 13B. Rank probabilities for fecal urgency at 3 months postoperatively. Supplementary Fig. 10B. Comparison-adjusted funnel plot for fecal urgency at 3 months postoperatively. Supplementary Fig. 11A. Network plot for use of antidiarrheal medication at 3 months postoperatively. Supplementary Table 14A. Relative effects table for use of antidiarrheal medication at 3 months postoperatively. Supplementary Table 14B. Rank probabilities for use of antidiarrheal medication at 3 months postoperatively. Supplementary Fig. 11B. Comparison-adjusted funnel plot for use of antidiarrheal medication at 3 months postoperatively. Supplementary Fig. 12A. Network plot for defecation frequency at 6 months postoperatively. Supplementary Table 15B. Rank probabilities for defecation frequency at 6 months postoperatively. Supplementary Fig .12B. Comparison-adjusted funnel plot for defecation frequency at 6 months postoperatively. Supplementary Fig. 13A. Network plot for fecal urgency at 6 months postoperatively. Supplementary Table 16A. Relative effects table for fecal urgency at 6 months postoperatively. Supplementary Table 16B. Rank probabilities for fecal urgency at 6 months postoperatively. Supplementary Fig. 13B. Comparison-adjusted funnel plot for fecal urgency at 6 months postoperatively. Supplementary Fig. 14A. Network plot for incomplete defecation at 6 months postoperatively. Supplementary Table 17A. Relative effects table for incomplete defecation at 6 months postoperatively. Supplementary Table 17B. Rank probabilities for incomplete defecation at 6 months postoperatively. Supplementary Fig. 14B. Comparison-adjusted funnel plot for incomplete defecation at 6 months postoperatively. Supplementary Fig. 15A. Network plot for use of antidiarrheal medication at 6 months postoperatively. Supplementary Table 18A. Relative effects table for use of antidiarrheal medication at 6 months postoperatively. Supplementary Table 18B. Rank probabilities for use of antidiarrheal medication at 6 months postoperatively. Supplementary Fig. 15B. Comparison-adjusted funnel plot for use of antidiarrheal medication at 6 months postoperatively. Supplementary Fig. 16A. Network plot for defecation frequency at 12 months postoperatively. Supplementary Table 19B. Rank probabilities for defecation frequency at 12 months postoperatively. Supplementary Fig. 16B. Comparison-adjusted funnel plot for defecation frequency at 12 months postoperatively. Supplementary Fig. 17A. Network plot for fecal urgency at 12 months postoperatively. Supplementary Table 20A. Relative effects table for fecal urgency at 12 months postoperatively. Supplementary Table 20B. Rank probabilities for fecal urgency at 12 months postoperatively. Supplementary Fig. 17B. Comparison-adjusted funnel plot for fecal urgency at 12 months postoperatively. Supplementary Fig. 18A. Network plot for incomplete defecation at 12 months postoperatively. Supplementary Table 21A. Relative effects table for incomplete defecation at 12 months postoperatively. Supplementary Table 21B. Rank probabilities for incomplete defecation at 12 months postoperatively. Supplementary Fig. 18B. Comparison-adjusted funnel plot for incomplete defecation at 12 months postoperatively. Supplementary Fig. 19A. Network plot for use of antidiarrheal medication at 12 months postoperatively. Supplementary Table 22A. Relative effects table for use of antidiarrheal medication at 12 months postoperatively. Supplementary Table 22B. Rank probabilities for use of antidiarrheal medication at 12 months postoperatively. Supplementary Fig. 19B. Comparison-adjusted funnel plot for use of antidiarrheal medication at 12 months postoperatively. Supplementary Fig. 20A. Network plot for defecation frequency at 24 months postoperatively. Supplementary Table 23B. Rank probabilities for defecation frequency at 24 months postoperatively. Supplementary Fig. 20B. Comparison-adjusted funnel plot for defecation frequency at 24 months postoperatively. Supplementary Fig. 21A. Network plot for use of antidiarrheal medication at 24 months postoperatively. Supplementary Table 24A. Relative effects table for use of antidiarrheal medication at 24 months postoperatively. Supplementary Table 24B. Rank probabilities for use of antidiarrheal medication at 24 months postoperatively. Supplementary Fig. 21B. Comparison-adjusted funnel plot for use of antidiarrheal medication at 24 months postoperatively. Supplementary Table 25A. Relative effects table for postoperative anastomotic leakage in the sensitivity analysis. Supplementary Table 25B. Rank probabilities for postoperative anastomotic leakage in the sensitivity analysis.