Ketamine has been increasingly used to treat mental health conditions yet there is a lack of safety data on intramuscular (IM) and sublingual (SL) dosing in a community setting. The Roots to Thrive Ketamine assisted Therapy (RTT-KaT) program is a 12-week program with 12 Community of Practice (CoP) group therapy sessions and three ketamine sessions.To provide preliminary data on RTT-KAT adverse events to subsequently inform safe use of IM and SL ketamine for the treatment of psychiatric disorders.Retrospective chart review of the RTT-KaT Program on four cohorts (n=128) between September 2020 to December 2021. Eligible patients include those with post-traumatic stress disorder, depression, generalized anxiety, burnout/adjustment disorder, substance use disorder, obsessive compulsive disorder, disordered eating, and disordered sleep. Baseline characteristics and adverse events were captured including medication administration before, during, and after RTT-KaT sessions. Chi-squared test with Yates’ continuity correction was used to assess side effects in subgroups from ketamine administration.RTT-KaT was well tolerated with no loss to follow up. There were 351 IM (mean dose = 102.553mg) and 96 SL (mean dose = 276.667mg) sessions of ketamine. Of the 448 sessions, the prevalence of elevated blood pressure increased by 12.31% from baseline (36.85%), with all post-treatment elevations being transient. The prevalence of elevated blood pressure post-KaT session was also similar between IM (+11.69% from 37.71% baseline) and SL (+15.12% from 32.98% baseline) administration. Regarding adverse effects, 12.05% of sessions experienced nausea , 2.52% had an episode of vomiting , 3.35% had a headache , and seven sessions experienced dizziness. The incidence of adverse events was not significantly associated with past psychedelic experiences , nor past psychiatric diagnosis . . There was no significant association between administration route and incidence of nausea, which was the most common side effect. Male gender was also significantly associated with lower incidence of nausea .The group therapy model described provides a comprehensive approach and presents a promising model for operating a KaT program outside of a clinical trial setting.These findings suggest good safety and acceptability for RTT-KaT among individuals seeking treatment for mental health issues. Majority of participants did not experience adverse reactions and the adverse events that were recorded involved transient symptoms that were resolved with rest and/or medications.None Declared


Blepharoglossum is a rare orchid genus of the Malaxidinae primarily distributed in tropical Pacific islands, with several species occurring in the Taiwan and Hainan Islands of China. Currently, the monophyletic status of Blepharoglossum has been challenged, and the phylogenetic relationships among its allied groups have remained unresolved with traditional DNA markers. In this study, we initially sequenced and annotated the chloroplast (cp) genomes of two Blepharoglossum species, Blepharoglossum elegans (Lindl.) L. Li and Blepharoglossum grossum (Rchb.f.) L. Li. These cp genomes of Blepharoglossum share the typical quadripartite and circular structure. Each of the genomes encodes a total of 133 functional genes, including 87 protein-coding genes (CDS), 38 tRNA genes and 8 rRNA genes. By comparing the sequence differences between these two cp genomes, it was found that they are relatively conserved in terms of overall gene content and gene arrangement. However, a total of 684 SNPs and 2664 indels were still identified, with ycf1, clpP, and trnK-UUU protein-coding genes having the highest number of SNPs and indels. In further comparative analyses among the six cp genomes in Malaxidinae, significant sequence divergences were identified in the intergenic regions, namely rps16–trnQ-UUG, trnS-GCU–trnG-GCC, rpoB–trnC-GCA, trnE-UUC–trnT-GGU, trnF-GAA–trnV-UAC, atpB–rbcL, petA–psbJ, psbE–petL, psbB–psbT, trnN-GUU–rpl32, trnV-GAC–rps7, and rps7–trnL-CAA, and five coding regions, including matK, and rpoC2, ycf1, and two ycf2 genes. Phylogenetic analysis indicated that Blepharoglossum and Oberonia form a highly supported sister group relationship. Our results are consistent with previous studies and present increased resolution among major clades. Blepharoglossum (Schltr.) L. Li of Malaxidinae , includes species previously ascribed to Liparis Rich. s.l. or Cestichis Thouars ex Pfitzer [Cestichs and allied genera using ITS and matK sequence data [Blepharoglossum was placed as sister to Oberonia Lindl. with weak support. Based on morphological characters, they observed a clear difference with regard to their rachis characters. Blepharoglossum species have terete rachises; in contrast, species of Cestichis and Stichorkis Thouars possess slightly or strongly flattened rachises. In addition, Blepharoglossum species have a deeply bilobed lip apex with margins that are lobulated and ciliolate to deeply lacerate. Based on these findings, Blepharoglossum was elevated to full generic status. Blepharoglossum comprises approximately 39 species that are mostly distributed in tropical Asia–Pacific islands, including Peninsular Malaysia, Borneo, Sumatra, Java, Sulawesi, the Philippines, Papua New Guinea, New Caledonia, and Fiji, with some species found in Myanmar, Thailand, Vietnam, and Taiwan and Hainan Islands in China [The orchid genus  Pfitzer ,2. The mnce data . Blepharin China .Blepharoglossum is a precious orchid genus, whose species are all listed in Appendix I of the Convention on International Trade in Endangered Species Wild Fauna and Flora (CITES). Due to the destruction of its habitat and people’s enthusiasm for the mining of orchid plants, it is further lacking resources. In Flora of China, the Blepharoglossum encompasses 5 distinct species, namely B. condylobulbon (Rchb.f.) L. Li, B. elegans (Lindl.) L. Li, B. fissilabrum (T. Tang and F.T. Wang) L. Li, B. grossum (Rchb.f.) L. Li, and B. latifolium (Lindl.) L. Li. Despite the fact that they serve as an important resource for orchids, the cp genomes of these five species have not been previously characterized. Therefore, it is imperative to conduct a comprehensive analysis to explore the sequence variability within the cp genome of this genus, as it holds significant implications for conservation efforts. Moreover, there is still uncertainty regarding the intergeneric relationships within Malaxidinae at present. Although the Blepharoglossum was previously and for a long time placed as Liparis s.l. or Cestichis due to the resemblances in floral morphology, the molecular systematics and morphology evidence indicated that the Blepharoglossum clade and other Liparis alliances had distant relationships and significantly different characteristics. Thus, it had been separated from epiphytic Liparis s.l. and elevated to a generic status in recent years [Blepharoglossum and the phylogenetic relationships within its relevant groups have been controversial due to their limited resolution with traditional sequences from one or a few markers in previous studies that await stronger evidence to support them. This is probably because its relatives have a notoriously complicated taxonomy. Therefore, the development of more effective genetic resources on the basis of high-resolution molecular markers is necessary for further phylogenetic studies of Blepharoglossum.The nt years . NeverthmatK. However, the cp genome data have demonstrated remarkable advantages in resolving the developmental relationships and systematic positions of complex taxa, resulting in significant improvements in accuracy and reliability [The chloroplast (cp) genomes have been extensively employed in various areas of research, such as in species identification, evolutionary assessments, and in the development of molecular markers ,6,7. Resiability ,11. Moreiability ,13.Blepharoglossum species, B. elegans and B. grossum, which have not been previously characterized. We then analyzed their differences and compared sequence features with those of four other species in allied genera within Malaxidinae. The aims of this investigation were two-fold: (1) to elucidate and contrast the cp genomes of Blepharoglossum, identifying variations among these six species, and (2) to reconstruct a robust phylogenetic tree of Blepharoglossum and its closely related taxa using cp genome sequences, thereby elucidating their phylogenetic relationships and verifying their taxonomic status within Malaxidinae. Our cp genome data presented here will provide an informative and valuable genomic resource for facilitating species authentication and evolutionary analysis in the Orchidaceae.In this study, we sequenced and assembled the complete cp genomes of two B. elegans and B. grossum were obtained from the living collections of the greenhouse of South China Botanical Garden, Chinese Academy of Sciences , which were introduced from the field populations in Hainan, China in 2021. The voucher specimens had been lodged at the herbarium of South China Botanical Garden (IBSC), under collection numbers YWT042 (B. grossum) and YWT043 (B. elegans). Total genomic DNA was extracted from fresh leaf tissues using a Plant Genomic DNA kit  following the manufacturer’s instructions.The samples of The total DNA was fragmented and used to set up short-insert libraries and the qualified libraries were sequenced with PE150 bp on the DNBseq-2000RS platform at Beijing Genomics Institute . There was ~3.0 Gb total amount of data and about 20× sequencing depth. Then we used SOAPnuke software  to filteBlepharoglossum from NCBI GenBank database as reference sequences for assembly, i.e., Liparis auriculata Blume ex Miq. (GenBank accession no.: MN200365), Liparis bootanensis Griff. (GenBank accession no.: MN627759), Liparis nervosa (Thunb.) Lindl. (GenBank accession no.: MN641753), and Liparis pingtaoi  J.M.H. Shaw (GenBank accession no.: MN627758). The assembled cp genomes were annotated with PGA [Blepharoglossum species under analysis, using B. grossum as a reference sequence.Clean data were used for assembling the complete cp genomes by using GetOrganelle v1.6.2e . We downwith PGA , then mawith PGA . UtiliziBlepharoglossum species noted above, we downloaded four published sequences of their allies from the NCBI database, i.e., L. bootanensis (GenBank accession no.: MN627759), L. viridiflora (Blume) Lindl. (GenBank accession no.: MW691170), Stichorkis gibbosa (Finet) J.J. Wood (GenBank accession no.: OM759993), and Oberonia seidenfadenii (H.J. Su) Ormerod (GenBank accession no.: MN414241) in order to identify the genetic and genomic features among different genomes. The location of genes on the IR/SC boundaries were visualized by IRscope [O. seidenfadenii selected as a reference [In addition to the newly sequenced genomes of two  IRscope . The diveference .Blepharoglossum and related genera, we reconstructed the phylogenetic tree of Malaxidinae based on 25 cp genome data representing 24 species. In addition to the 2 newly sequenced Blepharoglossum cp genomes, another 23 cp genomes were obtained from the NCBI GenBank database. Sequences of three species, i.e., Epipactis purpurata Sm., Neottia japonica (Blume) Szlach., and Cephalanthera humilis X.H. Jin, were selected as outgroups based on earlier phylogenetic studies [To gain a more thorough understanding of the intergeneric relationships between  studies . All the studies . The besB. elegans and B. grossum that were obtained in this study have been deposited in the NCBI GenBank database under the accession numbers OP859143 and OP859144, respectively. Both Blepharoglossum cp genomes exhibited the typical circular and quadripartite structure characteristic of most angiosperms  and 157,549 bp (B. grossum) in size. The cp genome of B. elegans was extremely similar to that of B. grossum. The length of the LSC regions are 84,984 bp (B. elegans) and 85,588 bp (B. grossum), the SSC regions are 17,731 bp (B. elegans) and 17,765 bp (B. grossum), and the IR regions are 27,049 bp (B. elegans) and 27,098 bp (B. grossum), respectively  and indels (insertions/deletions) were identified with the cp genome of B. grossum as a reference. Compared to the B. grossum reference genome, 684 SNPs and 2664 indels were detected in B. elegans . In all these cp genomes, the rpl22 gene crossed the LSC/IRb border. The IRa/LSC junction was located between rps19 and psbA, and variation between psbA and the Ira/LSC border ranged from 85 bp to 185 bp , with the fragments varying from 1055 bp to 1159 bp in the IRb region. In contrast, the ycf1 gene of O. seidenfadenii was nearly completely situated in the SSC region, extending only 2 bp to the IR region, and thus, no pseudogene was created at the border. Furthermore, overlaps between the ψycf1 fragment and the ndhF gene were detected at the IRb/SSC junction. In B. elegans, B. grossum, and S. gibbosa, the ndhF gene was located 65 bp–68 bp away from IRb/SSC, whereas in L. bootanensis and L. viridiflora, it was only 9 bp away, which also had a 9 bp overlap with ψycf1.The IR/SC boundary regions and adjacent genes were compared among the chloroplast genomes of o 185 bp . HoweverndhF gene has been pseudogenized or lost in most orchid cp genomes, which is correlated with shifts in the position of the junction of the IR/SC regions [O. seidenfadenii was considerably shorter. The IR regions of O. seidenfadenii was only 24,278 bp in length, while the IR regions of the other species examined were much longer, approximately 27,000 bp in length  as a reference. Our analyses showed that higher divergences were distributed in the non-coding regions compared to the protein-coding regions. Expectedly, significant divergences were identified within non-coding intergenic spacer (IGS) regions. Additionally, for both coding and non-coding regions, the LSC and SSC regions exhibited higher sequence variations than the two IR regions. Notably, we found two separate deletions of about 240 bp in the psbE–petL region and 473 bp in the trnE-UUC–trnT-GGU region for both of B. elegans and B. grossum cp genomes. In addition, B. elegans carried a unique deletion of 400 bp in the ycf4–cemA intergenic region. Most of the genetic variabilities were identified in the intergenic spacers, such as rps16–trnQ-UUG, trnS-GCU–trnG-GCC, rpoB–trnC-GCA, trnE-UUC–trnT-GGU, trnF-GAA–trnV-UAC, atpB–rbcL, petA–psbJ, psbE–petL, psbB–psbT, trnN-GUU–rpl32, trnV-GAC–rps7, and rps7–trnL-CAA. Somewhat higher sequence variations in the coding regions were observed, including rpoC2, ycf1, and two ycf2 genes  that have evolved independently.Overall, the topology of the phylogenetic tree inferred from our maximum likelihood (ML) analyses was largely consistent with those from previous studies using molecular data from a few markers of nuclear and plastid regions ,31,32; hBlepharoglossum species. A total of 4 genes  exhibited more than 20 SNPs, while 6 genes  had indel numbers exceeding 20, which suggests that these genes could serve as promising molecular markers for studying Blepharoglossum, aiding in practical applications such as resource conservation and species identification. We also offered information on the sequence divergences in these cp genomes and the four other published Malaxidinae cp genomes. Among these 6 cp genomes, genomic divergences were detected as promising DNA barcodes, including 12 intergenic regions and 5 protein-coding genes. In particular, two separate deletions occurred in the two Blepharoglossum cp genomes which were not found in the other allies. The positions of the IR/SSC boundaries associated with the ycf1 genes and the ndhF genes were quite variable for these six cp genomes. Additionally, the phylogeny inferred from the complete cp genomes greatly improved the phylogenetic resolution of the Stichorkis and Blepharoglossum clades and will likely contribute to our knowledge of the generic circumscription within Malaxidinae.This study first shed light on the structure and content of the complete chloroplast genomes of two


Enterococcus faecium is a common pathogen responsible for urinary tract infections (UTIs) and often establishes extensive colonization within the intestinal tract. Our aim was to assess the genomic and transcriptomic differences between colonized E. faecium without UTI (only-colonization) and colonized E. faecium causing UTI (endogenous infections).We investigated the correlation between fecal isolates from the same patient and UTI-causing isolates using PFGE and WGS, and classified fecal isolates into two groups: those that solely colonized and those associated with endogenous urinary tract infections. We characterized the genomes of colonization-only and endogenously infected isolates by Scoary GWAS, and the transcriptomes of the isolates at 3 h urine exposure to assess pathogen-related changes.E. faecium and nine isolates of only-colonized E. faecium were characterized and carbon and nitrogen regulated metabolisms such as genes encoding the phosphotransferase (PTS) system were enriched in endogenously infected E. faecium. Transcriptome analysis revealed significant differences in gene expression in the PTS system, lysine synthesis, galactose metabolism and citrate import between endogenously infected and only-colonized E. faecium isolates, highlighting the important role of certain carbon regulatory genes in the colonization and survival of endogenously infected E. faecium.Based on PFGE and WGS, eight isolates of endogenously infected In only-colonized and endogenously infected isolates, we observed differential expression patterns of genes related to carbon metabolism and amino acids, suggesting that metabolic diversity is a strategy for isolates leading to endogenous infection. Enterococcus faecium is a common cause of urinary tract infections. Because drug resistance is prone to establish an advantage of intestinal colonization, we isolated and performed homology analysis of intestinal colonized E. faecium and urinary tract infections with E. faecium and analyzed the advantage of E. faecium leading to endogenous infection using genomic and transcriptomics. We identified the carbohydrate and amino acid metabolic profiles of E. faecium that occur endogenously to provide an advantage for metabolic shifts. Our study provides insights for the control of clinical E. faecium infections.1.Urinary tract infections (UTIs) are one of the most common infections both within the community and in hospitals, which affect millions of people annually resulting in a significant burden on society . In the Enterococci are part of the intestinal microbiota and are also a common UTI causing pathogen. A decade-long study indicates that Enterococcus faecium is the fourth most common pathogen causing urinary tract infections and is the primary pathogen in neonatal urinary tract infections  with those of E. faecium isolates with endogenous infection . Our study will provide insights into endogenous infections of E. faecium.To our knowledge, the factors leading to endogenous infection with 2.2.1.E. faecium from the urinary and intestinal tracts were screened in our LIS system from the same patients, and eligible bacteria were thawed and isolated in our bacterial bank. In our study, a total of 49 strains of E. faecium were included. Bacteria were stored in strain preservation solution at −80°C. We used MALDI-TOF/MS to determine the identity of the isolates. The isolates were named PX-YY-GI/U, where PX is the patient, YY is the isolate ID, GI is the gastrointestinal isolate, and U is the urinary tract infection isolate.This retrospective study was conducted at The First Hospital Affiliated to Zhejiang University School of Medicine. From January 2018 to December 2020, 2.2.Enterococcus faecalis ATCC29212 was used for quality control.Minimum inhibitory concentrations (MIC) were determined according to drug susceptibility guidelines established by the American Clinical and Laboratory Standards Institute (CLSI). The agar dilution method or broth dilution method was used to assess MIC for penicillin, ampicillin, vancomycin, teicoplanin, erythromycin, tetracycline, ciprofloxacin, levofloxacin, rifampicin, linezolid, and tigecycline, daptomycin. 2.3.E. faecium, and the assay was performed as described previously with slight modifications  to extract genomic DNA from all E. faecium isolates, purified DNA was sent to Whole-genome sequencing (WGS). WGS was performed using an Illumina HiSeq 2,500 instrument . Raw sequence reads were quality controlled using FastQC v.0.11.5 and trimmed using Trimmomatic v.0.40. Trimmed reads were assembled using SPAdes v.3.6. All assembled genome annotations were carried out using Prokka v.1.14.6. Roary v.3.12.0 was used for pan-genomic analysis of the annotated genomes, and Scoary v.1.6.16 was used to assess the association between the presence and absence of genes in UTIs. All genomes were analyzed for antibiotic resistance genes and virulence genes using Abricate v.1.0.1. Core-gene single-nucleotide polymorphisms (SNPs) were extracted by default parameters in snippy v.4.6.0. Putative duplicate regions, mobile genetic elements and recombination in the files were filtered using Gubbins v.2.3.4, an SNP-based maximum likelihood tree was inferred by FastTree v.2.1.5 and visualized using the ITOL web server. Multi-locus sequence typing (MLST) was performed using MLST v.2.22.0.We used the FastDNA2.5.Enterococci and shaken overnight at 200 rpm until the optical density (OD) at 600 nm was 0.4. After obtaining informed consent from each volunteer, urine was collected from healthy males and healthy females, and the urine was immediately filtered twice using a 0.22 μm membrane and stored at −80°C. Urine from three volunteers was then heated to 37°C and mixed, then the logarithmic growth phase Enterococci were exposed to urine for 3 h. After exposure, the bacteria were centrifuged, washed with PBS, and immediately preserved in liquid nitrogen.Bacteria were exposed to urine as described previously . Briefly2.6.®Plus Mini Kit (QIAGEN). RNA-seq was performed on a HiSeq 2,500 instrument from Meiji Biotechnology Inc. . Raw datas were quality checked using FastQC v.0.11.5, and low-quality reads were trimmed using Trimmomatic v.0.40. The filtered data were mapped to the assembled genome using Hista2 v. 2.2.1, and hit counts for encoding genes were generated using the featucounts command in Rsubread v. 2.8.1. Differential expression between control and experimental groups was detected by Deseq2, and those with adjusted p-values less than 0.01, Log2-fold change (Log2FC) greater than 1 were classified as differentially expressed genes (DEG). DEG was extracted for gene-ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.We extracted bacterial RNA using the Rneasy3.3.1.E. faecium species between gastrointestinal colonization and urinary tract infection (UTI) patients, we included as many E. faecium strains meeting the screening criteria as possible in our bacterial repository. We isolated a total of 49 E. faecium strains from 14 UTI patients, with 22 of them being colonization strains. In our cohort, E. faecium isolates belonged to the multilocus sequence typing (ST) ST78 (n = 32), ST = 555 (n = 9), which is a well-known multidrug-resistant and hospital-adapted spectrum in China, ST78 caused the infection in 11 patients. Analysis revealed that isolates from 8 infected patients were closely related genetically to previously colonized isolates, defining them as endogenous infections. Infections from the same patient had almost no single nucleotide polymorphism (SNP) differences , and this high-resolution genomic evidence suggests that UTIs caused by E. faecium are induced by colonization strains from the same patient. The colonization isolates from 6 patients showed no correlation with urinary isolates . Resistance genes for aminoglycosides, tetracyclines, lincomycin, and peptides were detected in the isolates, and the infecting E. faecium isolates from the same patient’s colonization exhibited similar resistance genes and resistance phenotypes, as shown in Due to the possible differences in 3.2.Based on PFGE and WGS, the intestinal colonized isolates were further differentiated into eight endogenously infected isolates  and nine only-colonized isolates , ISL3 family transposase ISEfa11 (group_2760), L-glyceraldehyde 3-phosphate reductase (gpr_2), ISL3 family transposase IS1476 (group_362), replication initiation protein (repN), IS3 family transposase ISPa31 , IS3 family transposase ISSde10 (group_1472), and fructosamine deglycase (frlB). Further, we identified two genes (rpiR and gltC), encoding the helix-turn-helix (HTH)-type transcription factor, enriched in only-GI isolates, which were present in only one endogenously infected isolates. Importantly, a critical gene involved in the regulation of virulence metabolism is rpiR, while gltC is involved in activating expression of the glutamine-2-oxoglutarate aminotransferase gene for glutamate production. These results suggest that endogenously infected E. faecium isolates have a specific and nutritional/environmental adaptive gene profile, which distinguishes them from colonized isolates.profiles , and we p values . Scoary 3.3.3.3.1.E. faecium transcriptomic results between BHI growth and 3 h after urine exposure. The global transcriptomes were determined for two isolates for both conditions, and significantly DEGs were identified.One endogenously infected (p6-137-GI) isolate and one only-colonized (P6-133-GI) isolate were selected for this study. These were isolated from the same patient and both belong to ST78. Due to differences in the genomes of endogenously infected isolates and only-colonized isolates, we hypothesized that they may exhibit different transcriptional dynamics when infecting the urinary tract and that endogenously infected isolates show upregulation of genes implicated in pathogenicity. Thus, to maximize the possibility of discovering regulatory genes in UTIs, we utilized RNA-seq to compare p < 0.05 as the threshold of significance. Only-colonized E. faecium presented up-regulated expression of gene related to chromosome segregation and protein folding, as well as down-regulated expression of genes related to ion transmembrane transport, purine nucleoside metabolism, amino acid metabolism, and ATP metabolism , copper-exporting P-type ATPase B(copB), and copper chaperone (copZ). We observed the regulation of cellular Cu homeostasis by both isolates, thereby up-regulating the interaction of cytoplasmic copper chaperone and Cu-ATPase to deliver more copper into the ion permeation path. In addition, pur genes encode enzymes that are essential in the ab initio synthesis of purines, and all bacteria require purine nucleotides to complete essential metabolic processes such as DNA, RNA, and protein synthesis, and similar upregulated expression was observed in both isolates.The RNA-seq results demonstrate that only the isolates colonized and endogenously infected exhibit upregulation of identical genes after exposure to urine . Followi3.3.3.E. faecium isolates showed similar differences when transferred from the BHI to the urine, there were differences in carbon source metabolism and amino acid metabolism between only-colonized and endogenously infected isolates  involved in the formation of lysine precursors from aspartate semialdehyde via the diaminopyrimidine (DAP) pathway showed consistent up-regulation in endogenously infected isolates, However the lysine biosynthetic pathway was significantly down-regulated in the only-GI isolates after urine exposure. Further, marked differences in the metabolic lactose pathway were observed between the endogenously infected isolates and the only-colonized isolates. The lacEF gene was significantly up- and down-regulated in the urine-exposed endogenously infected isolates and in the only-colonized isolates, respectively. LacEF encodes for phosphoenolpyruvate (PEP)-dependent phosphotransferase, which transfers lactose to the extracellular bacterial environment and produces lactose-6-phosphate. Similarly, LacG, which encodes a phosphorylated-β-galactosidase that hydrolyzes to produce lactose-6-phosphate and glucose, was up-regulated in urine-exposed endogenously infected isolates. Further, compared to BHI, endogenously infected isolates also up-regulated the tagatose-6-phosphate pathway-related operons (lacABCD) responsible for galactose-6-phosphate catabolism following urine exposure. Moreover, only-colonized isolates up-regulated the Leloir pathway-related operons . In addition, cit-encoding genes that mediate citrate transport and utilization were significantly expressed in endogenously infected isolates.In endogenously infected isolates, the 4.E. faecium is a significant causative pathogen for healthcare-associated infections, colonizing the intestines and easily infecting the urinary tract. Here, we describe comparative genomics and transcriptomics to identify and characterize specific genetic factors and adaptations of E. faecium from enteric colonization and UTI pathogenic colonization. Our findings highlight that  according to transcriptomic results upon exposure to urine. Through GO and KEGG analyses, DEGs are generally classified into expected categories transitioning from nutrient-rich to nutrient-poor living environments. The phosphotransferase system (PTS) regulatory system includes a large gene network associated with many metabolic processes and adaptive responses, including lactose/melibiose and mannitol PTS systems. PTS is one way bacteria use for sugar uptake, where the energy source is phosphoenolpyruvate. It is involved in the transport of various sugars into bacteria, including glucose, mannitol, fructose, and disaccharides, and its activation in different environments may vary, reflecting the appropriate carbon sources available in the environment. Furthermore, extensive research has shown that PTS regulation is associated with more complex genes that affect bacterial pathogenic mechanisms, including virulence and bacterial chemotaxis towards host cells  leads to the transcriptional inhibition of enzymes or genes involved in the utilization of other substrates when a nutrient source (such as glucose) is used . Howeverbstrates , 4. Our E. faecium. In our study, genes encoding citrate transport and metabolism were upregulated in E. faecium after exposure to urine. This was primarily achieved through membrane transport, followed by citrate lyase cleaving citrate into acetyl coenzyme A and oxaloacetate, which is then decarboxylated into pyruvic acid, sustaining the survival of E. faecium. Previous research has shown that the utilization of citrates promotes biofilm formation in Staphylococcus aureus  can be found in the article/supplementary material.GH: Writing – original draft. YZ: Writing – review & editing. HC: Data curation, Methodology, Writing – review & editing. TL: Methodology, Formal analysis, Writing – review & editing. LZ: Resources, Software, Project administration, Writing – review & editing. CL: Resources, Visualization, Data curation, Writing – review & editing. YC: Funding acquisition, Supervision, Validation, Writing – review & editing.


Bacillus amyloliquefaciens, a Gram-positive bacterium, has emerged as a versatile microorganism with significant applications in various fields, including industry, medicine, and agriculture. This comprehensive review aims to provide an in-depth understanding of the characteristics, genetic tools, and metabolic capabilities of B. amyloliquefaciens, while highlighting its potential as a chassis cell for synthetic biology, metabolic engineering, and protein expression. We discuss the bacterium’s role in the production of chemicals, enzymes, and other industrial bioproducts, as well as its applications in medicine, such as combating infectious diseases and promoting gut health. In agriculture, B. amyloliquefaciens has demonstrated potential as a biofertilizer, biocontrol agent, and stress tolerance enhancer for various crops. Despite its numerous promising applications, B. amyloliquefaciens remains less studied than its Gram-negative counterpart, Escherichia coli. This review emphasizes the need for further research and development of advanced engineering techniques and genetic editing technologies tailored for B. amyloliquefaciens, ultimately unlocking its full potential in scientific and industrial contexts. Bacillus amyloliquefaciens, a ubiquitous Gram-positive, aerobic bacterium, is commonly found in soil environments. This versatile organism has been utilized to produce a diverse array of heterologous proteins, including β-glucanases, acid-stable alpha-amylase, mesophilic alpha-amylase, cellulase, acid-soluble proteins, keratinase, and alkaline protease [B. amyloliquefaciens a valuable host for synthesising therapeutic proteins and industrially relevant enzymes. Furthermore, B. amyloliquefaciens offers several advantages for applications in agricultural biotechnology. It produces secondary metabolites that exhibit antimicrobial activities against many phytopathogenic microorganisms, promoting plant growth and enhancing overall plant health [B. amyloliquefaciens as a promising candidate for developing sustainable and eco-friendly agricultural practices.protease ,2,3. Thet health . These aBacillus amyloliquefaciens possesses remarkable physiological characteristics and a highly adaptable metabolism, enabling its cultivation of cost-effective media [Saccharomyces cerevisiae [B. amyloliquefaciens also benefits from robust expression systems with excellent genetic stability and lacks strong codon preferences [ve media . This barevisiae . B. amylferences .B. amyloliquefaciens has been established as an industrially significant bacterium in the production of biological indicators for sterilization and in biodefense research [research . The uniBacillus amyloliquefaciens. These tools range from classical counter-selection marker strategies to the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)—based genetic toolkits. The diverse protein secretion systems, along with the novel artificial promoter and ribosome binding site (RBS) libraries, further facilitate the production of extracellular enzymes [Over the years, extensive research has led to the development of various genetic modification tools for  enzymes ,11,12,13B. amyloliquefaciens is an ideal multifunctional probiotic, exhibiting significant potential in inhibiting pathogenic bacterial growth and enhancing nutrient assimilation. This microorganism is known to produce a wide array of enzymes, including α-amylase, protease, lipase, cellulase, xylanase, pectinase, aminotransferase, barnase, peroxidase, glucanase, and chitinase [B. amyloliquefaciens showed an important role in protecting against phytopathogenic fungi [hitinase ,14. BamHhitinase . The β-1ic fungi .B. amyloliquefaciens, combined with well-developed gene manipulation tools, enables the reconstruction of its cellular metabolism. The availability of public knockout collections further enhances its attractiveness as a host for metabolic engineering applications.The inherent genetic background of Bacillus amyloliquefaciens can significantly enhance the carbon content in compost, thereby improving soil quality and promoting crop growth [B. amyloliquefaciens is among the most prevalent bacteria known to colonize plants endophytically, playing a crucial role in the biocontrol of vascular plant pathogens [In the agricultural sector, research has demonstrated that adding an appropriate quantity of p growth . Moreoveathogens .B. amyloliquefaciens is also capable of forming complex biofilms, which can serve as living biological materials to produce various functional biomaterials. These include surface growth factors, antibiotics, lysozyme, and antimicrobial peptides for medical applications. The development of biofilms by Bacillus amyloliquefaciens enhances barley’s resistance to salt stress [B. amyloliquefaciens, such as the absence of efficient genetic editing systems, unclear transcriptional regulation of heterologous proteins, and restricted secretion of heterologous proteins [t stress . Howeverproteins .B. amyloliquefaciens [Most research has focused on optimizing factors such as signal peptides, transport channel levels, chaperone protein levels, and promoters in the expression and transport systems to enhance heterologous protein production in efaciens . NeverthBacillus amyloliquefaciens. Additionally, we have examined the factors that impede the broader utilization of this microorganism and discussed the underlying reasons. This comprehensive overview serves as a valuable resource for researchers seeking to gain a thorough understanding of B. amyloliquefaciens and its myriad applications , which has been manipulated using innovative plasmid designs for extrachromosomal and intrachromosomal purposes [ications ,8,10,21.purposes .amyE gene is important because it includes homologous sequences necessary for integration into the B. amyloliquefaciens chromosome. This integration can serve as a model for evaluating the success of different genetic engineering strategies: the amyE gene’s ability to integrate into the B. amyloliquefaciens chromosome can be a practical tool for checking and measuring the performance of various engineering strategies. For instance, if a modification to the amyE gene successfully integrates and improves the bacterium’s performance, the applied strategy was effective [B. amyloliquefaciens and shown the potential for further optimization and industrial utilization [The ffective . Previoulization ,13,22,23lization ,25,26,27lization ,29 has fBacillus amyloliquefaciens LB1ba02. By employing a single-plasmid CRISPR/Cas9n system, the researchers achieved an impressive 93% knockout efficiency for a single gene and simultaneous editing of three loci with 53.3% efficiency using a base editing CRISPR/Cas9n-AID system [aprE, nprE, wprA, and bamHIR), showcasing its potential as a rapid gene knockout and integration tool for B. amyloliquefaciens LB1ba02.Xin et al. (2022) recently reported the development of a CRISPR-based genetic toolkit designed for efficient gene editing, knockout, and integration in Bacillus amyloliquefaciens 205 [Bacillus [B. amyloliquefaciens 205, thereby facilitating the genetic manipulation of this bacterium. The toolbox also incorporated functional CRISPR systems, which provided precise editing capabilities to further manipulate the genetic material. This innovative strategy, particularly the implementation of interspecific transformation, holds potential applications across a broad range of Bacillus species. It allows for the transfer of beneficial traits from one species to another, broadening the possibilities for genetic enhancement in this bacterial genus [In a unique study, Zhao et al. (2020) developed a novel genetic toolbox to augment the endogenous expression of the mesophilic α-amylase gene in iens 205 . A key cBacillus . This meal genus .Bacillus species [The researchers applied several genetic engineering techniques to systematically increase gene expression levels. This included sporulation suppression, a technique that limits the bacteria’s natural process of forming spores, which allows more resources to be dedicated to the expression of the target gene. Additionally, transcript activation techniques were used to increase the production of the specific protein from its corresponding gene, and plasmid-based gene overexpression was utilized to boost the quantity of the target protein being produced. These strategic modifications can be applied across many  species .B. amyloliquefaciens strains by overexpressing the master regulator ComK from Bacillus subtilis (ComKBsu). Utilizing direct transformation of PCR-generated deletion cassettes, they executed tasks such as replicative plasmid distribution and gene knockout. Artificial induction of genetic competence in B. amyloliquefaciens strains carrying the plasmid pUBXC can be achieved through the overexpression of ComKBsu [Furthermore, Chen et al. (2016) successfully promoted spontaneous genetic competence in isolated  ComKBsu .Bacillus amyloliquefaciens, researchers employed genetic engineering techniques to improve the production of the alkaline protease BSP-1. They cloned the bsp-1 gene from a Bacillus subtilis strain and introduced it into B. amyloliquefaciens. The results demonstrated that the recombinant strain produced a higher quantity of alkaline protease than the wild-type strain, highlighting a potential approach for genetically engineering B. amyloliquefaciens to increase the production of novel alkaline proteases [In a study focused on enhancing the alkaline protease production capacity of roteases .B. amyloliquefaciens to enhance its antifungal properties and boost the production of eco-friendly antifungal lipopeptides. The study identified several genetic modifications that improved the engineered strains’ antifungal activity and lipopeptide production. These modifications included the deletion of genes involved in branched-chain amino acid biosynthesis and the overexpression of genes associated with lipopeptide synthesis. These findings suggest that genetic engineering can progressively enhance B. amyloliquefaciens’ antifungal capabilities, thereby facilitating the development of sustainable and environmentally friendly antifungal agents [More recent research has directed efforts toward genetically modifying l agents .Bacillus spp. are frequently utilized in the production of commercial enzyme preparations. To accommodate the secretion requirements of diverse proteins, Bacillus spp. possess several promoters and plasmid expression systems. The three conventional protein secretion pathways in Bacillus spp. include the general protein secretion pathway (Sec), the twin-arginine translocation pathway (Tat), and the ATP-binding cassette (ABC) transporters [Owing to their remarkable capacity to express and secrete proteins, sporters ,32,33  successfully cloned and expressed the transglutaminase (TGase) gene from w yields .B. amyloliquefaciens TGase gene was fused to the prodomain of the Streptomyces caniferus protease. This prodomain is crucial in protein folding, ensuring the TGase enzyme is formed correctly. It also prevents the premature activation of TGase within the bacterial cell, which could disrupt normal cellular processes. To activate the enzyme once it is correctly folded and exported from the cell, the 3C protease gene was also incorporated in the plasmid, allowing for in vivo removal of the prodomain and subsequent activation of the enzyme. The bicistronic vector constructed in this way was then used to transform an E. coli strain ready for expression.To overcome these challenges, the authors constructed a plasmid in which the B. amyloliquefaciens TGase gene in E. coli. The purified enzyme demonstrated activity across various substrates, indicating potential applications in the food industry [The study reported a successful expression of the industry .B. amyloliquefaciens BLB369 is known to produce this enzyme. Researchers have sequenced, cloned, and effectively expressed the glu369 full-coding sequence of the endo-β-1,3-1,4-glucanase gene in Escherichia coli Top10. To simplify the purification process, the glu369 coding sequence was integrated into the pKJD4 vector. The resultant OmpA-His-Glu369 fusion protein incorporated the OmpA signal sequence for E. coli periplasmic targeting, followed by a 6xHistidine tag for purification purposes. Owing to these beneficial attributes, endo-β-1,3-1,4-glucanase holds significant potential for various biotechnological applications [Endo-β-1,3-1,4-glucanases, classified as glycoside hydrolases, play a crucial role in the enzymatic depolymerization of 1,3-1,4 β-glucans and exhibit antifungal properties. ications  , B. amyloliquefaciens EH-9 was co-cultivated with germinated rice seeds, and the resulting supernatant was rich in GABA. When this supernatant was applied topically to mice’s dorsal skin, it significantly enhanced type I collagen production. This increase in type I collagen synthesis is attributed to the presence of GABA, as demonstrated by the loss of effect upon knocking down GABA-A receptors in mice [γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, has been utilized in treating conditions such as anxiety and sleep disorders. GABA has also shown potential as an anti-aging factor. Although GABA has been used as a food supplement, its efficacy and precise mechanism warrant further research.  in mice .B. amyloliquefaciens to produce GABA-rich rice by inoculating grain seeds with the bacterium. Mice fed with B. amyloliquefaciens-inoculated rice exhibited increased serum levels of neuropeptide Y  and improved head dips. A head dip is observed when a mouse puts its head in holes when placed in a hole board test to test anxiety levels. The study suggests this effect is mediated by gastrointestinal GABA-B receptors and the vagus nerve [Huang et al., (2023) harnessed us nerve .Bacillus species, with diverse applications across various fields [B. amyloliquefaciens LL3 has demonstrated the ability to synthesize this polypeptide, the yield remains low. Gao et al. (2019) employed genetic and metabolic engineering techniques to enhance the γ-PGA yield in this glutamic acid-independent γ-PGA-producing bacterial strain. In their study, glutamate metabolism pathways were partially inhibited by deleting the fadR, lysC, aspB, pckA, proAB, rocG, and gudB genes. The promoter Pc 2up was also inserted into the icd gene to regulate NADPH, while the srf and itu operons were removed. These modifications resulted in the development of a new strain, B. amyloliquefaciens NK-A11, which exhibited the highest γ-PGA titer (7.53 g/L), representing a 2.05-fold increase compared to B. amyloliquefaciens LL3Δupp [B. amyloliquefaciens JX-6 showed high yields of γ-PGA from fermenting corn stoke and soybean meal, where the yield was 112.82 g/L [Poly-γ-glutamic acid (γ-PGA) is a biocompatible and biodegradable polypeptide primarily produced by s fields . Althoug LL3Δupp . Further2.82 g/L .Pectinases, enzymes employed in the food industry and other sectors, are typically produced by bacteria or fungi; however, bacterial-derived pectinases are often preferred due to the ease of fermentation and the availability of various techniques to increase yield.B. amyloliquefaciens TKU050 to produce pectinases through bioprocessing banana peels, a pectin-containing by-product. By employing banana peels as a carbon source for pectinase synthesis, the approach not only reduced production costs but also promoted environmentally friendly practices. Furthermore, the study suggested that B. amyloliquefaciens TKU050 pectinases could have potential applications in prebiotic production [B. amyloliquefaciens strain KUB29. By employing a two-stage fermentation strategy, the researchers identified several enzymes exhibiting activity across various temperatures and pH levels, thereby enhancing enzyme synthesis. These findings indicated that the KUB29 strain holds promise for various industrial biotechnology applications, particularly in biofuel production and other bioproducts, as its enzymes provide a cleaner source than traditional methods [A primary challenge in pectinase production is the associated cost. To address this issue, Doan et al. (2021) utilized oduction . Devaraj methods .B. amyloliquefaciens D1 strain was isolated from Morchella crassipes. This strain can produce high levels of neutral proteases purified using chromatography and ammonium sulfate precipitation. In this study, the purified protease was used in soybean milk fermentation as an example of an industrial application where they increased the release of essential amino acids, which could improve the nutritional value of the fermented soybean milk [In a recent study, a ean milk .B. amyloliquefaciens JFL21 showed great antifungal activity and antibacterial activity against foodborne Gram-positive and Gram-negative pathogens. Furthermore, antimicrobial compounds produced by the JFL21 were purified, named Anti-JFL21 and were found to be composed of different lipopeptides, including surfactin, fengycin and iturin. This study has found that Anti-JFL21 showed great antimicrobial activity even to the multi-drug resistant pathogens with low impact on the probiotics [In the food industry, multi-drug-resistant foodborne pathogens are a great concern. A study has demonstrated that fengycin produced by obiotics .B. amyloliquefaciens C5 pro-duced potent antimicrobial surfactant lipopeptides, namely surfactin and bacillomycin D. The growth and antimicrobial activity of this strain were optimized using grape seed flour substrate, which demonstrated optimal performance at 0.2% (w/v) [B. amyloliquefaciens, offering diverse applications in the petrochemical, agricultural, and medical sectors. Gao et al. (2022) successfully im-proved the yield of iturin A by enhancing the expression of genes associated with fatty acid biosynthesis, resulting in the development of a new strain, B. amyloliquefaciens HZ-ADFTL2. This strain exhibited an increased iturin A yield of 2.96 g/L, representing a 6.59-fold enhancement compared to the original strain, B. amyloliquefaciens HZ-12 [B. amyloliquefaciens fmb-60 was enhanced with the addition of fructose to the culture me-dia. A subsequent study revealed that the increased fengycin synthesis in B. amyloliquefa-ciens fmb-60 was attributable to the upregulation of transcriptional factor sigmaH and two-component system degU and degQ, while downregulating the inhibitory regulator AbrB. Additionally, numerous genes involved in amino acid metabolism, fatty acid me-tabolism, and energy metabolism were upregulated or downregulated [B. amyloliquefaciens. They utilized B. amyloliquefaciens WH1 as a wild-type strain and knocked out several genes, including bdh to block the carbon over-flow metabolic pathway, kinA to disrupt bacterial sporulation and extend the production period, dhbF to enhance the availability of amino acids and fatty acids, and rapA to in-crease Spo0A~P levels. The quadruple knockout strain ΔkinAΔbdhΔdhbFΔrapA exhibited enhanced antifungal activity, and after fermentation, iturin and fengycin titers in the bio-reactor increased to 123.5 mg/L (a 22-fold increase) and 1200.8 mg/L (a 15.9-fold increase), respectively [B. amyloliquefaciens.Soussi et al. (2019) conducted a study illustrating that 2% (w/v) . Iturin ns HZ-12 . In a stegulated . Wang etectively . Table 1B. amyloliquefaciens SC06 has demonstrated a protective effect against high-fat diet (HFD)-induced obesity in animal models. Wang et al. (2019) found that mice fed an HFD combined with SC06 exhibited reduced fat accumulation, lower obesity levels, and a slight decrease in insulin resistance. Additionally, SC06 treatment provided liver protection and decreased the secretion of serum inflammatory factors, specifically IL-6, TNF-α, and leptin. The study also revealed that SC06 improved the intestinal microbiota composition, contributing to the observed protective effects [ effects .B. amyloliquefaciens on neuronal activity, microbiome composition, and glucose metabolism in an ischemic stroke model. In their study, Mongolian gerbils were fed chungkookjang, a soybean product fermented with B. amyloliquefaciens SRCM100730 (CKJ730) and B. amyloliquefaciens SRCM100731 (CKJ731), before inducing ischemic stroke via artery occlusion. The results indicated that the treatment reduced neuronal cell death, ameliorated stroke-related neurological symptoms, enhanced glucose metabolism by preserving β-cells in the pancreas, and improved insulin sensitivity. Furthermore, the treatment exhibited an anti-inflammatory effect, as evidenced by the reduced serum levels of proinflammatory cytokines TNF-α and IL-1β, while preserving the gut microbiome constituents, particularly Bacteroidia and Clostridia [Jeong et al. (2020) investigated the impact of ostridia .Bacillus amyloliquefaciens strain, X030, has been identified to produce a lipopeptide called bacillomycin Lb, which exhibits anticancer potential against various cancer cell lines [B. amyloliquefaciens X030 improved the microbiome composition in grass carp by promoting the presence of probiotics and reducing opportunistic bacteria in the intestine. The strain was also found to produce macrolactin A, an antibacterial agent that collectively enhanced grass carp resistance to certain infections. Furthermore, grass carp fed with B. amyloliquefaciens X030 exhibited increased expression of IL-8 (a chemokine), C3 (involved in liver stress tolerance), and IgM, thereby improving the immunity of grass carp against Aeromonas hydrophila X040 and A. veronii X005 infections [A novel ll lines . Additioll lines . A studyfections .Acinetobacter spp. is a nosocomial pathogen known for forming robust biofilms and exhibiting increasing antibiotic resistance, making the treatment of infections caused by this pathogen challenging. Al-Dulaimi et al., (2021) conducted a preliminary study evaluating the potential use of a probiotic strain, B. amyloliquefaciens B-1895 cell-free supernatant (CFS), against Acinetobacter spp. isolates from selected clinical cases. CFS’s antimicrobial and anti-biofilm activities were tested alone or in combination with polymyxin E. The study demonstrated that at high concentrations, CFS of B. amyloliquefaciens B-1895 inhibited both the growth of Acinetobacter spp. and biofilm formation. The combination of polymyxin E and CFS exhibited a synergistic effect, with lower minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) values compared to each agent used individually [In hospital settings, vidually .B. amyloliquefaciens also plays a significant role in animal health. In a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis model in mice, camel milk enriched with B. amyloliquefaciens PBT-3 (BEY) demonstrated beneficial effects [ effects . BEY tre effects .Bacillus amyloliquefaciens BLCC1-0238, either alone or in conjunction with mannan-oligosaccharides (MOS), led to a decrease in mortality rates, reduced intestinal mucosal lesions, and improved growth performance. This outcome appears to be associated with the elevated expression of claudin-3 and peroxisome proliferator-activated receptor-gamma coactivator-1α in both pretreatment groups. Furthermore, the addition of MOS to the bacterial treatment resulted in upregulation of mucin-2 [Bacillus spp. led to an increased incidence of intestinal inflammation due to alterations in the gut microbiome. Additionally, a decline in fertility was observed [B. amyloliquefaciens.In a comprehensive investigation of subclinical necrotic enteritis in broiler chickens, researchers observed that pretreatment with  mucin-2 . In a seobserved . Table 2Bacillus amyloliquefaciens, when isolated from the rhizosphere, has exhibited notable antifungal and antibacterial properties and the ability to promote plant growth, making it a valuable resource in agriculture. Plant growth-promoting rhizobacteria (PGPR) are recognized for their capacity to safeguard plants from biotic and abiotic stress, enhance plant growth, and increase crop yields . Wu et al. (2019) demonstrated that both diffusible and volatile organic compounds (VOCs) produced by B. amyloliquefaciens L3 exhibited antifungal activity, particularly against Fon while promoting watermelon growth. The study identified 2-nonanone and 2-heptanone as the most potent antifungal VOCs, with acetoin and 2,3-butanediol acting as growth promoters in a dose-dependent manner [B. amyloliquefaciens DHA55 displayed antifungal activity against Fon and other phytopathogenic fungi, mediated by the production of lipopeptides such as iturin, surfactin, and fungycin [t manner . Additiofungycin .B. amyloliquefaciens LZN01 against Fon was attributed to the production of myriocin, which disrupts fungal growth at the cellular level. The antifungal mechanism appears to involve myriocin’s impact on the expression of numerous membrane-related genes, primarily affecting sphingolipid metabolism, steroid biosynthesis, and glycerophospholipid metabolism. Additionally, myriocin disturbs protein processing in the endoplasmic reticulum and influences ABC transporters. These effects lead to decreased membrane fluidity and compromised cell membrane integrity, resulting in shrinkage and eventual death of Fon cells [B. amyloliquefaciens subsp. plantarum FZB42 (isolate DMK-7-2) demonstrated antifungal activity against Fon and promoted watermelon growth [In a separate study, the antifungal effect of on cells . Similarn growth .Bacillus amyloliquefaciens B1408 has demonstrated enhanced cucumber growth and biocontrol effects against Fusarium oxysporum f.sp. cucumerinum (Foc), which causes Fusarium wilt in cucumber plants. The beneficial effects of B. amyloliquefaciens B1408 appear to be mediated by inducing abnormalities, deformation, and cytoplasmic extravasation in Foc hyphal morphology. Additionally, B1408 alters the rhizosphere microbial community, providing protection against Fusarium wilt [B. amyloliquefaciens B9601-Y2 was found to promote maize seedling growth, improve soil quality by enhancing enzyme activity, increase chlorophyll content, and reduce the infection index by antagonizing Bipolaris maydis, which causes southern corn leaf blight [Similarly, ium wilt . In anotf blight .B. amyloliquefaciens S1 exhibits biocontrol effects against Clavibacter michiganensis, the causative agent of bacterial canker in tomato plants, when tested under net house conditions. The biocontrol effect is attributed to the production of siderophores, lytic enzymes, and antimicrobial metabolites. Moreover, in vitro experiments demonstrated that B. amyloliquefaciens S1 possesses significant phosphorus solubilization and indole acetic acid production capabilities [Gautam et al. (2019) reported that bilities . In a saB. amyloliquefaciens AH2 as a fungicide on grapes. The fungicidal effect of B. amyloliquefaciens AH2 was deemed effective against grey mold, the target pathogen. Furthermore, the toxicity of B. amyloliquefaciens AH2 was assessed in workers handling the bacterium, with no reported side effects or hypersensitivity reactions. However, a few individuals experienced allergic reactions to the bacterium’s metabolites. In mammalian studies, no adverse effects were observed, and the bacterium did not cause infectious disease in rats. However, the toxic effects of the bacterium’s metabolites remain inconclusive, necessitating further assessment. As a result, the study could not exclude the possibility of toxic effects from the bacterium’s products or metabolites [Anastassiadou et al. (2020) evaluated the use of abolites .Bacillus amyloliquefaciens SQR9, are more effective in treating saline soil and restoring balance to enhance crop yield [B. amyloliquefaciens SQR9 may involve increased total soluble sugar content, decreased reactive oxygen species, and reduced sodium ion levels, ultimately restoring sodium balance in the soil [B. amyloliquefaciens is effective in countering heat stress in plants [Bacillus amyloliquefaciens NBRI-SN13 inoculation on rice cv. Saryu-52 seedlings subjected to various abiotic stresses  and phytohormone treatments, it was found that B. amyloliquefaciens NBRI-SN13 promoted proline and total soluble sugar (TSS) production and increased the expression of late embryogenesis abundant (LEA) and dehydrin (DHN) genes, all of which are associated with enhanced stress tolerance. Additionally, SN13 upregulated the expression of glutathione S-transferase (GSTs), an important antioxidant enzyme [Plant growth-promoting rhizobacteria (PGPR) has been demonstrated to mitigate soil salinization, an abiotic stressor  that negatively impacts crop growth and yield by reducing agricultural land availability . Researcop yield . The potthe soil . Furthern plants . In a stt enzyme .B. amyloliquefaciens, which led to oxidative stress, damage to cell wall components, and negative impacts on energy metabolism and cell growth. However, co-exposure to copper ions did not exhibit any synergistic effects. The study demonstrated that the bacterium resisted the toxicity of PLA MPs through sporulation [Li et al. (2023) raised concerns regarding the toxic effects of polylactic acid microplastic (PLA MPs) exposure on rulation .Bacillus amyloliquefaciens in response to acidic environments as defined by the pH scale [B. amyloliquefaciens adjust to acidic environments [Recent research has been conducted to investigate the adaptation mechanisms of pH scale . The sturonments .B. amyloliquefaciens in agriculture, food production, and environmental management.Understanding these adaptation mechanisms may be valuable in developing strategies to mitigate the negative effects of acid stress on bacterial survival and growth, ultimately benefiting various applications of Bacillus amyloliquefaciens possesses a comprehensive array of genetic tools, promoters, and plasmid expression systems, making it a valuable resource in synthetic biology, metabolic engineering, and protein expression. Furthermore, it can potentially produce chemicals, enzymes, and other industrial bioproducts. This review discussed the advantages of B. amyloliquefaciens as a chassis cell in terms of genetic engineering, heterologous gene expression, and its applications in industry, medicine, and agriculture. Despite its numerous promising applications, B. amyloliquefaciens remains less studied than its Gram-negative counterpart, Escherichia coli, even in the context of advanced methodologies and rapid tool development.The Gram-positive bacterium B. amyloliquefaciens is its less efficient plasmid synthesis than E. coli. To address this issue, future research could focus on developing modified B. amyloliquefaciens strains that enable direct plasmid synthesis. Conversely, its high recombination rate offers certain advantages for the development of genome editing tools. B. amyloliquefaciens has already been established as a significant platform strain for the commercial production of various chemicals and enzymes. The availability of advanced engineering techniques and cutting-edge genetic editing technologies for controlling and regulating metabolic pathways further enhances the potential of Bacillus strains as exceptional industrial production hosts.One primary barrier to the widespread adoption of Bacillus amyloliquefaciens, enabling them to leverage its properties for various applications and research endeavors. The continuous advancement of fundamental and applied studies further solidifies the prominent position of B. amyloliquefaciens as one of the leading microbes in the fermentation industry. We anticipate that this evaluation will aid metabolic engineers in effectively harnessing established engineering approaches to guide metabolic engineering efforts for optimizing B. amyloliquefaciens cellular factories. Moreover, we hope that increased emphasis will be placed on fostering the development of metabolic engineering strategies and techniques specifically tailored for B. amyloliquefaciens.This review aimed to provide readers with a comprehensive understanding of the characteristics of B. amyloliquefaciens, a greater variety of technologies, tools, and methodologies will be employed in the future, enhancing its potential for application in both scientific and industrial contexts.As more scientists and engineers contribute to research on


Trypanosoma brucei and several other microbial pathogens, virulence genes involved in antigenic variation, a key pathogenesis mechanism essential for host immune evasion and long-term infections, are located at subtelomeres, and expression and switching of these major surface antigens are regulated by telomere proteins and the telomere structure. Therefore, understanding telomere maintenance mechanisms and how these pathogens achieve a balance between stability and plasticity at telomere/subtelomere will help develop better means to eradicate human diseases caused by these pathogens. Telomere replication faces several challenges, and the “end replication problem” is a key obstacle that can cause progressive telomere shortening in proliferating cells. To overcome this challenge, most eukaryotes use telomerase to extend the G-rich telomere strand. In addition, a number of telomere proteins use sophisticated mechanisms to coordinate the telomerase-mediated de novo telomere G-strand synthesis and the telomere C-strand fill-in, which has been extensively studied in mammalian cells. However, we recently discovered that trypanosomes lack many telomere proteins identified in its mammalian host that are critical for telomere end processing. Rather, T. brucei uses a unique DNA polymerase, PolIE that belongs to the DNA polymerase A family (E. coli DNA PolI family), to coordinate the telomere G- and C-strand syntheses. In this review, I will first briefly summarize current understanding of telomere end processing in mammals. Subsequently, I will describe PolIE-mediated coordination of telomere G- and C-strand synthesis in T. brucei and implication of this recent discovery.Telomere maintenance is essential for genome integrity and chromosome stability in eukaryotic cells harboring linear chromosomes, as telomere forms a specialized structure to mask the natural chromosome ends from DNA damage repair machineries and to prevent nucleolytic degradation of the telomeric DNA. In  VSG swiepressed . VSG swisequence . DNA douwitching , as homowitching , and HDR. brucei . Consistwitching . Furtherwitching . In P. felomeres . In addifamilies , and thifamilies . Neverthfamilies . For exawitching . Therefowitching . Interesde novo, it poses another problem. Telomerase uses single-stranded DNA with G-rich sequence as its substrate  but not NHEJ  (TbRAP1)  to the telomere to facilitate telomeric repeat replication as its mammalian homologs , which is essential for the full-level expression of the active VSG   while TbRAP1 does not bind TERRA  , a RAP1 (TbRAP1) , and TbTian TIN2    and telo (TERRA) . TERRA itelomere , and it telomere , while Rtelomere . Indeed,ing rate . Therefoing rate . In contnd TERRA . TbTIF2 vicinity , and TbTct roles , althougT. brucei that do not seem to have any homologs in mammals   . In addiexamined . Furtherexamined . These sre ssDNA , and TbR by PICh , suggest complex . HoweverT. brucei telomere chromatin  by RNAi within 24 h, and PolIE-depleted cells quickly stop proliferating, indicating that PolIE is essential for l growth . Howeverl growth . Neverthl growth . Depletil growth . Furtherl growth .T. brucei telomeres have the expected terminal telomere 3′ overhang structure, but this overhang appears to be very short (∼12 nt in asynchronized cells)  , suggestd cells) , and lond cells) . Howevertructure , indicattructure . It is ktructure , althougtructure   has also been identified from all three proteomic approaches described above aimed to identify telomere proteins (T. brucei. PPL2 is essential and has translesion DNA polymerase activities that catalyzes error-prone bypass of 6-4 photoproduct, but it lacks a primase activity (Interestingly, proteins , stronglactivity . In addiactivity . These oT. brucei to its mammalian host. However, T. brucei telomere protein complex has several distinct components than the human telomere complex. Even though TbTRF, TbRAP1, and TbTIF2 have telomere functions that are more comparable to their mammalian homologs, the sequence homology between these proteins and their mammalian counterparts is only limited within a few functional domains (T. brucei telomere proteins play essential roles in telomere maintenance, and further investigation will help us better understand how different eukaryotes use different players to achieve the same goals.In summary, telomere end processing  appears  domains . In addiT. brucei telomere maintenance mechanisms suggest that TRF, RAP1, and TIN2 homologs have been evolved much earlier than TPP1/POT1 and CST homologs (T. brucei is a type A DNA polymerase that arose and diversified in the kinetoplastids lineage (Kinetoplastids are ancient organisms diverged from the mammalian branch in the evolutionary tree more than 500 million years ago. Recent studies on homologs . Specifi lineage . WhetherT. brucei undergoes antigenic variation. Hence, vaccination is not effective, and chemotherapy is a key approach for treating African trypanosomiasis. Although subtelomere plasticity facilitates antigenic variation, telomere stability is critical for genome integrity and parasite proliferation. As T. brucei clearly uses distinct and essential telomere factors than its mammalian host for telomere end processing, the T. brucei-unique telomere proteins are promising targets for anti-trypanosome agents. Better understanding about the degree of effects of telomere plasticity and stability on VSG switching will further help us identify anti-parasite means that have clear detrimental effects on parasite growth without enhancing antigenic variation efficiency inadvertently. In addition, T. cruzi and Leishmania are kinetoplastid parasites that cause devastating human diseases affecting more than ten million people worldwide. They are closely related to T. brucei, and homologs of all essential telomere proteins are present in T. cruzi and Leishmania. It is expected that telomere maintenance mechanisms among these three trypanosomes are highly conserved, and knowledge gleaned from T. brucei on telomere protein functions and telomere end processing would also help eradication of T. cruzi and Leishmania.


Toxoplasma gondii is a worldwide distributed zoonotic protozoan capable of infecting a wide range of mammals (including humans) and birds as intermediate hosts. Migratory wild birds, through interconnecting countries along their flyways, can play a role in the spatial spread of T. gondii and could contribute to its sylvatic cycle. Additionally, hunted wild birds used for meat consumption could represent a further source of human infection. To determine the presence of T. gondii in wild birds, a total of 50 individuals belonging to the Anseriformes and Charadriiformes orders were sampled during the 2021–2022 hunting season in Northern Italy. Cardiac muscle samples of three Northern shovelers (Anas clypeata), two wild mallards (A. platyrhynchos), one Eurasian teal (A. crecca), and one Northern lapwing (Vanellus vanellus) were positive for the molecular detection of T. gondii based on a targeted amplification of the B1 gene. A 14% (7/50) overall positivity was observed in the sampled population. Results from this study suggest a moderate exposure of wild aquatic birds to T. gondii, highlighting the importance of a further characterization of T. gondii in its wildlife hosts. Toxoplasma gondii is a widespread zoonotic apicomplexan protozoan potentially able to infect all the warm-blooded animal species [T. gondii. The parasite can therefore be found in various environmental matrices where, upon sporulation, oocysts become infectious and can remain viable up to several years [T. gondii forms life-long persistent cysts located prevalently in neural and muscular tissues, such as brain, retina, and skeletal and cardiac muscles [T. gondii and the fecal excretion of its oocysts can only happen in the definitive hosts [T. gondii by influencing the survival time of unsporulated oocysts; furthermore, seasonal fluctuation in precipitation rates influence the dispersion of sporulated oocysts [ species . The lifal years . This enal years ; any waral years . For mamal years . When in muscles . Through muscles ,6. Neverve hosts . Environ oocysts .T. gondii in wildlife and humans can determine heterogeneous clinical observations. Toxoplasmosis can indeed be fatal or chronic, with disease severity affected by host-dependent or parasite-dependent variables  [Infections caused by ve dose) . In aviave dose) ,9,10.T. gondii, oocysts are exclusively deposited on land and can therefore disperse in freshwater following heavy rainfalls, owing to the hydrophilic nature of their surface [T. gondii infections in wild aquatic species. In aquatic environments, intermediate avian hosts belonging to different feeding groups  appear to be subjected to a similar infection probability [T. gondii [T. gondii infection in these animals suggests the presence of an oocysts-contaminated aquatic habitat. Furthermore, being huntable aquatic birds and a human food source, the consumption of raw or undercooked meat, especially derived from niche products, could represent an underappreciated source of Toxoplasma gondii infection [T. gondii in wild aquatic birds hunted in the wetlands of Northern Italy, where wintering migratory individuals from different breeding grounds congregate seasonally.Considering the terrestrial definitive host of  surface ,11; thisbability . In birdbability ,15,16,17. gondii ,19. Consnfection . Given thttps://www.izsvenezie.it/documenti/temi/influenza-aviaria/piani-sorveglianza/piano-nazionale-influenza-aviaria-2021.pdf, accessed on 22 October 2021) and the Commission Delegated Regulation (EU) 2020/689. As an inclusion criterion, only individuals whose entire carcass was preserved were included in the molecular survey hereby presented. Sampling activities were conducted from October 2021 to January 2022 in two hunting grounds (Spatula clypeata), 18 Eurasian teals (Anas crecca), 7 mallards (A. plathyrhynchos), 5 Northern lapwings (Vanellus vanellus), and 1 gadwall (Mareca strepera) were sampled. The sex and age class (juvenile of the year or adult) of each individual were recorded by trained ornithologists, as reported in A total of 50 hunted wild aquatic birds were included in this study, selected among 124 individuals sampled within the application of the National Avian Influenza (AI) Surveillance Plan 2021 ( grounds d of the ® Genomic DNA Mini kit (Invitrogen by Thermo Fisher), according to the manufacturer’s protocol. Nested PCR targeting the glycerol-3-phosphate dehydrogenase gene (B1) of T. gondii was performed with minor modifications of the protocol described by Jones et al. [T. gondii positive DNA was added as a positive control. PCR products were electrophoresed on 2% agarose gel stained with SYBR Safe DNA Gel Stain  in 0.5× TBE. For sequencing, the amplicons were excised and purified by Nucleo-Spin Gel and PCR Clean-up  and sequenced with an ABI 3730 DNA analyzer . Consensus sequences were compared with published sequences by a BLAST search .Heart tissue samples were collected and stored at −20 °C until processing. Genomic DNA was purified from 25 mg of tissue using the Pure Links et al. . Brieflyp value below 0.05 (p < 0.05) was considered significant.Data analyses were performed using GraphPad Prism (version 8.0) software . Fisher’s exact test (two sided) was used to determine the association between the molecular results and the species, age class, and sex of the birds tested. A T. gondii. Sequence data were submitted to the NCBI GenBank database under the following accession numbers: OQ646717-23. As shown in T. gondii and species (p > 0.1), sex (p = 0.43), or age class (p = 0.09) of the birds tested.The amplification of the B1 gene was successful in 7 out of the 50 heart specimens tested (14%) . BLAST sT. gondii oocysts as widely distributed in the environment, especially in aquatic habitats [T. gondii in hunted wild aquatic birds. The population tested included species with different habits and migratory strategies. As a result, parasitic DNA was found in 14% (7/50) of the heart samples tested, showing a moderate exposure of waterbirds to the infection.Considering habitats ,24, thisToxoplasma gondii molecular detection in individuals belonging to the Anseriformes order, namely, Northern shovelers, mallards, and Eurasian teals, confirmed previously published records for other geographic areas surveyed [T. gondii in different intermediate hosts, suggesting a possible local infection [T. gondii epidemiology. surveyed ,26,27,28surveyed . Epideminfection ,30,31. WT. gondii infection has already been reported by Nardoni et al. [T. gondii oocysts could be associated with inefficient sewage treatment, water discharge, and water runoff, as already demonstrated for marine environments [T. gondii detection in the studied population since mid-October, local exposure to the parasite may also be likely for the adult ducks. In fact, an experimental study in chickens reported the detection of tissue cysts in brain, heart, liver, spleen, or lungs, starting from 7–15 days post-infection [For Northern lapwings, a species belonging to the Charadriiformes order commonly found in open lands and mudflats, i et al.  in centri et al. ; it is tronments . Consideronments  and the nfection . T. gondii in wild birds have been carried out testing different matrices [T. gondii PCR detection, even in wild aquatic birds [T. gondii was molecularly detected in 3 out of 9 and 8 out of 12 individuals, respectively. However, as reported by Opsteegh et al. [T. gondii infection in cattle, also seronegative animals could harbor tissue cysts. The loss or absence of a detectable antibody titer in infected individuals has been observed in several bird species [T. gondii positive tissues, and seropositive animals with tissues negative for parasite DNA). The molecular screening of all the individuals studied, as performed in this survey, could therefore reflect the actual percentage of birds that harbor tissue cysts. Furthermore, although no correlation with sex and age was hereby observed, these variables deserve attention in further investigations with a larger sample size to obtain a better understanding of T. gondii epidemiology in wild aquatic birds. Molecular studies aimed at the detection of matrices ; in thisic birds ,25,26, aic birds ,38. Althic birds ,41,42,43h et al.  for T. g species ,46, whil species  found diT. gondii in heart samples may indicate the presence of tissue cysts in edible muscles, such as the pectoral ones, whose consumption could determine a meat-borne transmission of the parasite. In addition, it has been shown that handling wild or domestic animal carcasses without appropriate hygiene practices could lead to infection due to the accidental contamination of hand or other equipment from bradyzoites released from tissue cysts during cutting practices [From a One Health point of view, the detection of ractices ,48,49,50T. gondii in wild aquatic birds from Northern Italy, suggesting their importance as biological indicators for T. gondii contamination of aquatic habitats and their potential contribution to the sylvatic cycle of the parasite. Furthermore, migratory species could act as T. gondii long-distance transmitters, considering their ecology and habits. An additional genetic characterization of the positive samples is needed to establish the role of migratory birds in linking countries where different genotypes circulate.To conclude, results from this study highlight moderate exposure to


Mycobacteriumbovis (M. bovis) infections, the diagnosis of which is a challenging task that requires keen clinical awareness. We report the case of an adult male treated with BCG who presented with fever, nonspecific constitutional symptoms, hepatic cytolysis, and cholestasis. After a detailed workup, the diagnosis was made of disseminated M. bovis infection with hepatic, pulmonary, renal, and ureteral involvement. Prompt anti-tuberculosis treatment resulted in clinical and analytical improvement. This case highlights the importance of early recognition of this serious complication in patients with BCG exposure, as well as the difficulty in confirming the diagnosis for proper treatment.Intravesical instillation of bacillus Calmette-Guérin (BCG) is a crucial adjunctive therapy in the treatment of bladder carcinoma. Its systemic complications are rare but include disseminated  Mycobacterium bovis (M. bovis), is the recommended immunotherapy for all high-grade and some low-grade non-muscle-invasive bladder cancer (NMIBC) [Intravesical instillation of bacillus Calmette-Guérin (BCG), a live attenuated strain of  (NMIBC) . In pati (NMIBC) ,3. Howev (NMIBC) .M. bovis infection, persistent fever, and any organ involvement beyond the genitourinary system [BCG-induced complications may manifest with a spectrum of symptoms, ranging from self-limited irritative voiding symptoms to potentially severe sepsis . Less coy system . IdentifA 77-year-old man diagnosed with high-grade bladder urothelial carcinoma (pT1) was submitted to transurethral resection followed by immunotherapy, initially with BCG instillation, weekly for six weeks, followed by monthly for two months. A few days after the first monthly instillation, he presented with fever, severe fatigue, nausea, and anorexia. Due to the persistence of symptoms associated with weight loss, after the second monthly instillation, he went to the emergency department. On admission, he was febrile (38.3°C), with no other findings upon physical examination. Blood tests were performed and revealed mild anemia (hemoglobin (Hb) 12.5g/dL) with a normal white blood cell and platelet count, acute kidney injury (creatinine (Cr) 1.47mg/dL), increased levels of C-reactive protein (47.7mg/L), hepatic cytolysis, and cholestasis  without hyperbilirubinemia. He was then admitted to the Infectious Diseases Department for further investigation.M. bovis. A hepatic biopsy was also performed. Histology revealed granulomatous hepatitis with giant Langhans cells  and 65.5% in tissue biopsy, while the identification of granulomatous inflammation in specimens is available in 86.3% of cases [M. bovis infection and the difficulty in diagnosing it, given its nonspecific clinical manifestations and the potential involvement of multiple organs. Nevertheless, the possibility of this diagnosis must not be overlooked in a patient undergoing BCG treatment who exhibits systemic inflammation and shows clinical or analytical signs of organ dysfunction. The challenge stems from differentiating this condition from other inflammatory and infectious conditions. Achieving a diagnosis requires a comprehensive approach that involves obtaining biopsies from the affected organs and conducting culture tests to confirm the presence of an M. bovis infection. This approach allows for the prompt initiation of appropriate treatment while discontinuing intravesical instillation of BCG.This case highlights the importance of recognizing a disseminated