Helicobacter pylori (H. pylori) in human gastric mucosa is highly associated with the occurrence of gastritis, peptic ulcer, and gastric cancer. Antibiotics, including amoxicillin, clarithromycin, furazolidone, levofloxacin, metronidazole, and tetracycline, are commonly used and considered the major treatment regimens for H. pylori eradication, which is, however, becoming less effective by the increasing prevalence of H pylori resistance. Thus, it is urgent to understand the molecular mechanisms of H. pylori pathogenesis and develop alternative therapeutic strategies. In this review, we focus on the virulence factors for H. pylori colonization and survival within host gastric mucosa and the host antimicrobial responses against H. pylori infection. Moreover, we describe the current treatments for H. pylori eradication and provide some insights into new therapeutic strategies for H. pylori infection.The colonization of  Helicobacter pylori (H. pylori) is commonly identified in the stomach. It has been reported to infect more than a half of the world’s population and can persist life-long without eradication [H. pylori infection is thought to occur during childhood within families by the oral-oral or fecal-oral route, with a higher prevalence in developing countries probably due to poor hygiene and crowded conditions [H. pylori infection is a risk factor for chronic gastritis and peptic ulcer and recognized as a class I carcinogen of gastric cancer by the World Health Organization (WHO) [Gram-negative, microaerophilic, spiral-shaped dication ,2. The Hnditions . The H. on (WHO) .H. pylori are owing to the action of a variety of bacterial virulence factors. On the one hand, H. pylori can generate numerous ureases to neutralize the acidic environment of the stomach lumen. On the other hand, the bacterial flagellar-dependent motility enables H. pylori to penetrate the mucus layer toward the gastric epithelium, where the pH is almost neutral [H. pylori to the surface of gastric epithelial cells [H. pylori then produce numerous virulence factors, including two well-known cytotoxins-cytotoxic associated gene A (CagA) and vacuolating cytotoxin A (VacA), which can modulate the biological function of gastric epithelial cells and induce the release of proinflammatory cytokines to cause chronic inflammation [H. pylori was thought to be a non-invasive bacterium thar mainly lived in the mucus layer [H. pylori can invade and replicate in the intracellular compartments of different cell types [H. pylori, e.g., induction of antimicrobial peptides [The successful colonization and pathogenesis of  neutral . Moreoveal cells . The colammation . For a lus layer . Howeverll types , causingll types . Under tpeptides ,12, actipeptides , and incpeptides . H. pylori infection is an effective way to improve or resolve the associated pathology. In clinic, antibiotic-based therapies are usually recommended to treat H. pylori infection. However, treatment failure has been increasing in recent years due to poor patient compliance and antibiotic resistance [H. pylori in primary infections or after initial treatment failure. In this review, we aim to describe the interaction between host cells and H. pylori and detail the role of host cellular antimicrobial responses against H. pylori infection. Moreover, we discuss the current treatments for H. pylori infection and highlight key findings of novel alternative therapies, providing some novel insights on developing host cell antimicrobial responses as therapeutic strategies for H. pylori eradication.Eradication of sistance . TherefoH. pylori are associated with bacterial colonization and the development of gastroduodenal diseases. Here, we briefly introduce some well-known bacterial adhesins and cytotoxins which are involved in host cell-bacterial interaction and pathogenesis. The virulence factors of H. pylori to the gastric epithelium is not only crucial for successful colonization and pathogenesis but also essential for invasion into host cells. Numerous studies have revealed that H. pylori expresses a variety of adhesion factors that could bind to related cell surface molecules, such as sugars or proteins. More than 30 H. pylori outer membrane proteins (OMPs) have been identified, which play pivotal roles in bacterial attachment to the gastric mucosa. These OMPs could be divided into two groups: Hop (Helicobacter outer membrane proteins) and Hor (Hop-related proteins) subgroups [The adhesion of ubgroups ,17. BeloH. pylori. BabA mediates the adhesion of bacterium to the Lewis b blood group antigen (Leb), a major antigen expressed by gastric mucosa [H. pylori adherence to Leb on the gastric epithelium promoted type IV secretion system (T4SS) activity, resulting in the production of pro-inflammatory cytokines and other factors that contributed to the development of gastric tumorigenesis [Blood group antigen-binding adhesin A (BabA) is a major adhesin of c mucosa . BabA coc mucosa ,20,21. Iigenesis .H. pylori, could bind to sialic acid-modified glycosphingolipids, sialylated Lewis x and Lewis a (sLex and sLea) [H. pylori-infected patients but returned to normal after H. pylori eradication [b is thought to be the first step of early H. pylori infection, wherein H. pylori anchors to the gastric epithelial cell surface. The binding of SabA to sLex further strengthens the connection [H. pylori as escape mechanisms from host immune defense responses [Sialic acid-binding adherence (SabA), another well-known OMP of nd sLea) . The levnd sLea) . In addidication . Bindingnnection . Intriguesponses . Notablyesponses .H. pylori outer membrane protein, which promoted interleukin-8 (IL-8) secretion in a cagPAI-dependent manner [H. pylori to gastric epithelial cells, while the exact host receptor of OipA remains unknown [oipA of H. pylori was found to be associated with the occurrence of gastric cancer [The outer inflammatory protein A (OipA) was initially identified as a t manner . Subsequ unknown . The phac cancer . Furtherc cancer . H. pylori, is a 120–145-kDa immunogenic protein encoded in a 40-kb bacterial genomic DNA region known as cag pathogenicity island (cagPAI) [H. pylori to deliver macromolecules into other bacteria or cells [H. pylori can be further classified as CagA-positive and -negative strains [H. pylori strains isolated in Western countries  are CagA-negative which are less associated with the occurrence of peptic ulcer and gastric carcinogenesis, whereas almost all H. pylori strains isolated in East Asian countries  are CagA-positive with stronger pathogenicity [Cytotoxin-associated gene A (CagA), the most well-studied virulence factor of (cagPAI) . Presumaor cells . Based o strains . Approxigenicity . H. pylori could use T4SS apparatus to translocate CagA into host cells where the tyrosine within EPIYA motif of CagA are phosphorylated by c-Src and c-Abl tyrosine-protein kinases, resulting in disturbed cell signaling pathways and enhanced tumorigenesis [H. pylori CagA could induce epithelial-mesenchymal transition (EMT) in gastric cancer by activating YAP pathway [H. pylori promoted DNA damage in gastric cancer via downregulating DNA repair protein Rad51 [CagA shows a highly polymorphic Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region . Accumuligenesis ,39. In aigenesis . It is r pathway . Anotherin Rad51 .H. pylori gene VacA, which is characterized by its ability to form pores and cause vacuolation in cultured eukaryotic cells [Vacuolating cytotoxin (VacA) is a secreted toxin encoded by ic cells . VacA isic cells  , “m” region  and the intermediate region  determine the pore-forming ability and pathogenicity of VacA [All  of VacA . It has  of VacA ,54. InteH. pylori results in chronic inflammation and the related pathogenesis in stomach. Meanwhile, host cells also develop numerous antimicrobial responses to defend against H. pylori infection. Here, we describe some innate defense strategies exploited by host cells, especially for gastric epithelial cells and professional phagocytic cells, to fight against invading H. pylori, including induction of antimicrobial peptides, activation of cellular autophagy pathway, and increased oxidative stress.The successful colonization of H. pylori infection, human immune and epithelial cells can produce two major AMPs, cathelicidin/LL-37, and defensins, to protect the host against invading pathogens [Antimicrobial peptides (AMPs), also called host defense peptides (HDPs), are a kind of biologically active small peptides that are widely expressed in almost all living organisms and serve as an important part of the innate immune system to protect the host against a wide spectrum of pathogens . Most AMathogens . CAMP) gene encodes the peptide precursor human cationic antimicrobial peptide-18 (hCAP-18), which is then subject to C-terminal cleavage by extracellular serine proteinase-3 to generate active form of LL-37 [H. pylori infection can markedly induce both RNA and protein expression of LL-37 in the gastric epithelium in a T4SS-dependent manner [H. pylori infection [H. pylori and reduced H. pylori colonization in both in vitro gastric epithelial cell line model [As a part of the human innate immune system, cathelicidins are present in a variety of human tissues and organs . Circulaof LL-37 . LL-37 it manner . Mechaninfection . Activatne model  and in vne model . Due to ne model  and furtne model . In addine model ,68. H. pylori infection was reported to increase HNP1–3 levels in gastric juice and promote the release of active peptides from granulocytes. However, the exact mechanisms of H. pylori-induced α-defensin are still unclear [H. pylori infection [H. pylori-induced NLRC4 inflammasome was reported to activate IL-18 to inhibit HBD1 expression in an NF-κB-dependent manner [H. pylori. Mechanically, internalization of bacterial peptidoglycan caused by cagPAI is recognized by nucleotide-binding oligomerization domain-1 (NOD1), resulting in induction of HBD2 [H. pylori infection via a TAK1 (transforming growth factor β-activated kinase-1)-EGFR -p38α axis, which is dependent on the type IV secretion system but independent of CagA or NOD1 [H. pylori [Defensins are cysteine-rich cationic AMPs regarded as a part of host innate immune systems to fight against bacterial infection . α-defen unclear ,73. On t unclear . HBD1 isnfection ,75. Intrt manner . In cont of HBD2 . In addi or NOD1 . Moreove or NOD1 . Similar or NOD1 . Further. pylori . H. pylori could invade the gastric epithelial cells to cause persistent infection [H. pylori [Autophagy is thought to be the original form of innate immune response of eukaryotic cells against intracellular microorganisms . Besidesnfection ,82. The . pylori .H. pylori [“Autophagy” (the Greek word means “self-eating”) refers to cellular machinery that degrades unnecessary and dysfunctional intracellular components through a lysosome-dependent manner . It allo. pylori .H. pylori could invade and multiply in different cell types, such as gastric epithelial cells, macrophage, and dendritic cells [H. pylori could be engulfed by AGS cells through a zipper-like mechanism involving various cellular signal transduction pathways [H. pylori virulence factor VacA induced autophagy in a manner dependent on the channel-forming activity of VacA, but independent of urease, CagA, or type IV secretion system [H. pylori and RIP2 on early endosome to induce cellular autophagy and inflammation, and this effect was independent of VacA [H. pylori could exploit autophagy and hijack immature autophagosomes to generate a protected reservoir [H. pylori [Accumulating evidence suggests that ic cells ,87,88. Tpathways . Autophapathways . Terebizn system . Low-denn system . In addi of VacA . Althougeservoir ,93. Altheservoir . Capurro. pylori .H. pylori in gastric epithelium could induce chronic inflammatory response and recruit phagocytic cells, such as macrophages, neutrophils, and inflammatory monocytes. In phagocytes, NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS), respectively responsible for the generation of superoxide (O2−) and nitric oxide (NO), are induced to defend against invading pathogens. O2− and its derivatives, known as reactive oxygen species (ROS), as well as NO and its derived intermediates, called reactive nitrogen species (RNS), play important roles in host antimicrobial response [H. pylori infection, although how these changes contribute to H. pylori-mediated pathogenesis is still controversial [Colonization of response . In addioversial ,98,99.H. pylori lipopolysaccharide (LPS) was reported to activate the transcription of NOX1 in guinea pig gastric mucosal cells [2−) and hydrogen peroxide (H2O2) could directly damage bacterial DNA, lipids, amino acid resides, and Fe-S clusters [H. pylori induce ROS both in phagocytic cells and gastric epithelial cells, they could express superoxide dismutase (SOD) and catalase to protect themselves [The NOXs, which are widely expressed in most mammalian tissues, are responsible for the generation of ROS. al cells . In addial cells ,102,103.clusters . Althougemselves ,105.2−) to form oxidant peroxynitrite (ONOO−), which exhibits a more powerful effect to kill pathogens [H. pylori infection was reported to markedly induce iNOS expression in macrophage and gastric tissues through activating NF-κB signaling [H. pylori arginase could consume L-arginine, the substrate for iNOS, to inhibit NO production of activated host immune cells [H. pylori inhibited iNOS translation in macrophages to restrict host defense [The iNOS are another key enzyme expressed by host immune cells to convert L-arginine to NO through an oxidoreductase reaction . NO coulathogens . H. pyloignaling . Howeverne cells . In agre defense .H. pylori infection is highly associated with the occurrence of related gastric disorders. It is now generally accepted that all H. pylori infections should be eradicated unless there are compelling reasons. Different guidelines for the management of H. pylori-related diseases have been published in many regions. However, the challenge remains huge given the increasing resistance of H. pylori to current antibiotic-based treatments. We describe below some novel therapeutic strategies which shed light on the future regimens for H. pylori eradication [The dication ,111.H. pylori infection. It has been shown that a variety of antibiotics, including clarithromycin (CLR), amoxicillin (AMX), and metronidazole (MTZ), could effectively kill H. pylori in in vitro environment. However, almost the antibiotics can only work effectively in neutral or near neutral conditions, in contrast to the highly acidic environment of H. pylori-resided human stomach. This makes the delivery of active antibiotics more difficult. To solve this problem, a proton pump inhibitor (PPI) is taken together to inhibit the acid secretion by the stomach [H. pylori replicates when stomach pH is larger than 6 and becomes more susceptible to the antibiotic treatment [Antibiotic-based treatments are commonly used to eradicate  stomach . More imreatment . H. pylori infection [According to the latest version (2017) of the American College of Gastroenterology (ACG) clinical guideline, in the region with low clarithromycin resistance (<15%) rate, a 14-day course of triple therapy containing PPI, CLR, and AMX (or MTZ) is recommended. Patients with penicillin allergy should receive a 10- to 14-day course of bismuth quadruple therapy comprising PPI, bismuth, tetracycline, and a nitroimidazoles. Given the increasing prevalence of CLR resistance worldwide, alternative regimens have been proposed. One of the alternative regimens is 10- to 14-day sequential therapy with PPI and AMX for 5–7 days, followed by PPI, CLR and nitroimidazoles for the next 5–7 days. Another alternative regimen is non-bismuth quadruple (concomitant) therapy containing PPI, CLR, AMX, and nitroimidazoles. Besides, other regimens, including hybrid therapy  and levofloxacin-based therapy, were also proved to be effective in treating nfection ,114. Altnfection .H. pylori, while alleviating the antibiotic-associated adverse effects [Bacillus mesentericus + Clostridium butyricum + Streptococcus faecalis in a standard triple therapy showed optimal efficacy and safety to enhance the therapeutic effect [H. pylori infection. However, no effective vaccines have been applied in clinical practice so far, which is probably due to the high adaptability of H. pylori to human immune response and the high genetic diversity of this bacterium [Notably, recent studies have identified that the addition of probiotics in standard antibiotic therapy could enhance the eradication rate of  effects ,116. A mc effect . Moreoveacterium ,119. H pylori resistance to antibiotics is increasing rapidly worldwide, representing the main challenge in H. pylori infection treatment [H. pylori in gastric biopsies of patients likely confers resistance of H. pylori to antibiotics, acid environments, and host immune responses [H. pylori. Below, we describe some novel strategies based on modulation of host-bacteria interaction and host antimicrobial responses.The prevalence of reatment ,120. In esponses ,121 and esponses . DevelopH. pylori colonization, invasion, and pathogenesis. Therefore, anti-adhesion therapy is proposed as a novel strategy to control H. pylori infection [H. pylori could attach to host cells via bacterial adhesin SabA interacting with sialic acid on the cell surface [H. pylori by prolonging drug residence time and increasing drug concentration at infection sites [H. pylori (OM-NPs) to mimic intact bacteria and found that OM-NPs could compete with bacteria for the cell surface binding sites [H. pylori infection [Adhesion is a prerequisite for nfection . H. pylo surface . Numerouon sites . In adding sites . Moreovenfection ,127,128.H. pylori infection [H. pylori treatment. Zhou et al. found that Vitamin D3 (VitD3) could upregulate CAMP expression and inhibit H. pylori infection in mice, implying that the supplement of VitD3 might help control H. pylori infection. Mechanistically, VitD3 bound to vitamin D receptor (VDR) and facilitated its transport to the nucleus. The VitD3/VDR complex could bind to the promoter region of CAMP to promote its expression, leading to an enhanced antibacterial effect [H. pylori eradication [Human cathelicidin LL-37 peptide and defensins play a pivotal role in the innate defense responses against nfection ,64,71. Il effect . Besidesl effect . Given tdication .H. pylori can be considered a facultative intracellular bacterium because it can invade host cells and survival in immature autophagosomes [H. pylori in host cells plays a pivotal role in bacterial virulence and persistence and protects the bacteria from antibiotics [H. pylori virulence factor VacA could impair lysosomal function by targeting lysosomal Ca2+ channel TRPML1 protein to disrupt endolysosomal trafficking, thereby creating an intracellular reservoir [H. pylori. Hu et al. reported that VitD3 treatment promoted the nuclear translocation of PDIA3/STAT3 complex to induce TRPML3 transcription, leading to activated lysosomal function in normal gastric epithelial cells to kill H. pylori. Notably, this process is independent of LL-37 activation [H. pylori [H. pylori.agosomes . The intibiotics ,133. H. eservoir . Activattivation . Meanwhi. pylori . TherefoH. pylori infection in gastric mucosa recruits immune cells, especially neutrophils, which could release reactive oxygen species (ROS) and reactive nitrogen species (RNS) to kill bacteria [H. pylori infection could contribute to gastric carcinogenesis [H. pylori have evolved a variety of strategies to resist oxidative stress [H. pylori infection while avoiding damaging normal tissues. In this regard, Tan et al. synthesized a graphitic nanozyme with pH-responsive oxidase-like activities, which could only catalyze the production of ROS in gastric acidic conditions but not in neutral intestinal environments. Besides, the synthesized nanozyme contains a hydrophobic alkyl tail to enable specific targeting of H. pylori and generating of high-concentration ROS only around the bacterial membranes [H. pylori. HP1021 orphan response regulator was reported to be a redox switch of H. pylori to control bacterial response to oxidative stress. Targeting HP1021 could abolish the induction of oxidative stress-related gene expressions in H. pylori. Thus, HP1021 is a potential therapeutic target in the treatment of H. pylori infection [The bacteria . Howeverogenesis . Even woe stress . Therefoembranes . Anothernfection . H. pylori in human gastric mucosa is the main cause of gastritis, peptic ulcers, and gastric cancer [H. pylori could attach to the gastric epithelial cell surface through the interaction of bacterial outer membrane proteins, e.g., OMPs, BabA, SabA and OipA, with corresponding ligands on host cell membranes [H. pylori invasion into cells, which may be responsible for immune evasion and treatment failure of H. pylori infection [H. pylori through producing antimicrobial peptides (LL-37 and defensins), triggering autophagic degradation of bacteria, and releasing ROS and RNS. Nevertheless, H. pylori has also evolved a series of defense systems for their survival (The colonization of c cancer . Approxiembranes . Bacterinfection . Accordisurvival .H. pylori eradication. However, treatment failure is increasing because of antibiotic resistance. To avoid antibiotic abuse, development of non-antibiotic therapeutic strategies is urgent. Typing the bacterial susceptibility to antibiotics and determining OMPS and virulence factors would help in further individualization of the treatment. Besides, modulation of host antimicrobial responses is promising to fight against invading H. pylori. Recent studies reported that administration of VitD and HDACi was shown to increase antimicrobial peptide levels [H. pylori infection is another alternative therapeutic strategy. Lastly, therapeutic strategies to enhance ROS and RNS level in the region of H. pylori infection while abrogating its antioxidant response is a new weapon to kill invaded H. pylori [H. pylori infection and could be used together to enhance the efficacy of antibiotics-based regimens or used as alternative regimens when current first-line treatment has failed in the future. However, preclinical and clinical studies are required to assess their efficacy and adverse effects, and the cost and compliance of patients should also be considered.Currently, antibiotic-based therapies are the major regimens for e levels ,139. Note levels . In addie levels . Thus, a. pylori ,137. In


Bacteroides uniformis and Anaerotruncus sp. 1XD22-93 were enriched in Donor CKD, while Lactobacillus johnsonii and Lactobacillus intestinalis were reduced. In addition, the increased abundance of microbial functions included tryptophan metabolism and lysine degradation contributing to the accumulation of protein-bound uremic toxins (PBUTs) in Donor CKD. Genome analysis indicated that FMT successfully differentiated groups of gut microbes and altered specific gut microbiota after 1 week of treatment, with Bacteroides uniformis and Anaerotruncus sp. 1XD22-93 increasing in CKD/CKD group as well as Lactobacillus johnsonii and Lactobacillus intestinalis being improved in CKD/Sham group. In comparison to CKD group, substantial PBUT buildup and renal damage were observed in CKD/CKD. Interestingly, compared to CKD or CKD/CKD group, tryptophan metabolism and lysine degradation were efficiently suppressed in CKD/Sham group, while lysine biosynthesis was promoted. Therefore, FMT considerably reduced PBUTs accumulation. After FMT, PBUTs and renal function in CKD/Sham rats remained the same as in Sham group throughout the experimental period. In summary, FMT could delay the malignant development of CKD by modifying microbial amino acid metabolism through altering the microenvironment of intestinal flora, thereby providing a novel potential approach for treating CKD.Chronic kidney disease (CKD) is associated with gut microbiome dysbiosis, but the role of intestinal flora in CKD treatment remains to be elucidated. Fecal microbiota transplantation (FMT) can be utilized to re-establish healthy gut microbiota for a variety of diseases, which offers new insight for treating CKD. First, 5/6 nephrectomy rats (Donor CKD) and sham rats (Donor Sham) were used as donors for FMT, and fecal metagenome were analyzed to explore potential therapeutic targets. Then, to assess the effect of FMT on CKD, sterilized 1/2 nephrectomy rats were transplanted with fecal microbiota from Donor sham (CKD/Sham) or Donor CKD (CKD/CKD) rats, and 1/2 nephrectomy rats without FMT (CKD) or no nephrectomy (Sham) were used as model control or normal control. Results showed that  Chronic kidney disease (CKD) is estimated to affect one sixth of the population worldwide, which causes significant morbidity, mortality, and severe socio-economic burden . The damIn recent decades, a great deal of effort has focused on the differences in gut microbial composition between CKD patients and healthy controls, confirming the relationship between gut bacterial and PBUTs . To alleFecal microbiota transplantation (FMT), a way to reconstitute receptor gut microbiota by transplanting of stool from a volunteer , offers In this study, flora from healthy and severe CKD rats were transplanted into a rat model of antibiotic-treated mild CKD. By interaction analysis of fecal metagenomic and serum PBUTs metabolome, it was determined how gut microbiome affected the development of CKD by PBUTs and whether transplantation from healthy colonic bacteria delayed the onset of CKD progression.® (SD) IGS Rats  were given a standard diet and housed under standard conditions. Rats were acclimatized and fed for 7 days before performing the experiments described below.Animal experiments were approved by the Animal Care and Use Committee of China Agricultural University. A total of 35 male CDn = 10) and a sham-operated group . The 5/6 nephrectomy was a bilateral nephrectomy and was performed in two stages. Firstly, 2/3 of the left kidney was removed, and then the whole right kidney was removed. The sham-operated group was used as a control for the 5/6 nephrectomy group, which was performed twice as same as the 5/6 nephrectomy to exclude the interference of the laparotomy operation. After 10 months of CKD progression, half of the rats in the Donor CKD group died. The 5/6 nephrectomy rats developed severe CKD by this point. Thus, the remaining nephrectomy rats and sham rats were donors of gut microbiota for the following experiment  and allowed to defecate freely. Donor feces were obtained simultaneously from all donor rats in each group, rather than from individual rat. Only feces that were not contaminated with pee were collected from the two groups of rats. Two sets of fecal samples from donors were placed on ice during the collection. After being returned to the laboratory, a final dilution with sterile saline  is performed in a sterile setting. Finally, fecal suspensions were mixed with 10% sterile glycerol and stored at –80°C until further analyzed.n = 15) or sham surgery . After 1-week operation convalescent period, 10 rats in the 1/2 nephrectomy group were treated with a 3-week course of mixed antibiotics treatment  to deplete gut microbiota (n = 5) and the other received intestinal flora from Donor CKD . FMT was performed by gavage daily  for 3 weeks. The remaining 1/2 nephrectomy rats served as model control, while sham-operated rats served as healthy control for 1/2 nephrectomy rats , blood urea nitrogen (BUN), and PBUTs were measured. From the beginning to the end of FMT, serum renal function indicators were measured weekly. Each group of rats was fasted overnight prior to blood collection. Crea and BUN contents were evaluated using a BS-420 automated biochemical analyzer . Six main PBUTs in serum such as indoxyl sulfate (IS), p-cresyl sulfate (PCS), phenyl sulfate (PS), hippuric acid, trimethylamine N-oxide (TMAO), and phenylacetyl glycine, were measured at the experimental end point by Bruker RP-HPLC with a Waters HSS T3 column  . The quaIn this study, the gene abundance and functional pathways at the taxonomic level for every bacterial species were determined using metagenomic sequencing of all genes present in the gut microbiome. The raw data was analyzed by online platform.  .And then, the raw reads from metagenome sequencing were removed adaptor sequences, trimmed and quality-controlled to generate the clean reads using “fastp”  on the fe-value cutoff of 1e–5  and paraffin-embedded. Hematoxylin & eosin (H&E), Masson, and PASM stains were done using staining kits  after 5-μm thick slices were cut. After staining, the tissue structure was observed under a light microscope and recorded.Kidney protein was extracted using total protein extraction kit . The concentration of protein extract was determined using standard BCA method . Proteins were loaded onto 5–8% gradient SDS-PAGE gels  for electrophoresis and transferred to PVDF membranes . And then the membranes were treated overnight at 4°C with the primary antibody against NADPH oxidase . Next, membranes were washed and treated with secondary antibodies linked to horseradish peroxidase . Subsequently, enhanced chemiluminescence compounds were used to create signals .t-test and one-way ANOVA. Free online platform Majorbio Cloud1 was used for metagenome analysis. Microbiota data were analyzed and graphed by the non-parametric tests Kruskal-Wallis sum rank test and Wilcoxon rank sum test. P < 0.05 was considered statistically significant.Results were expressed as mean ± SEM, and data analysis was performed using SPSS 21.0 software. Differences between groups were analyzed by P < 0.05) (P < 0.05) (P < 0.05), suggesting greater renal oxidative damage  . Moreove < 0.05) . Patholo < 0.05) . Finallye damage . These oP < 0.05) , while Anaerotruncus sp. 1XD22-93 and Bacteroides uniformis were most enriched in Donor CKD (P < 0.05)  . Statist < 0.05) . Taken tP < 0.05) with renal failure in Donor CKD group  through analysis of two donor groups  (P < 0.05) (P < 0.05), whereas lysine degradation was higher in Donor CKD group (P < 0.05). These results suggested that lysine metabolism, similar to tryptophan metabolism, played an equally important role in CKD. Taken together, these data indicate that variations in PBUTs are closely related to changes in amino acid metabolism caused by differences in intestinal flora.Major PBUTs were accumulated and showed a significant fold increase (KD group . A stronr groups . The KEG= 0.022) . Results= 0.022) . Based o < 0.05) . The mor < 0.05) . Lysine P < 0.05) after 1 week of FMT   in healthy rats was significantly negative correlated with PBUTs (P < 0.05)  (P < 0.05) (PBUTs in each group were at different level in the end of study . PBUTs l= 0.004) . The cor < 0.05) . In cont < 0.05) . Thus, P < 0.05) . At the  < 0.05) . In summP < 0.05), and the residual kidney exhibited hyperplasia (P < 0.05) (P < 0.05)  . One wee < 0.05) . Interes < 0.05) . In agre < 0.05) . Moreove < 0.05) . In geneIt is becoming interesting that the interaction between host and its gut microbiota has important effect on the health of host . DiffereAnaerotruncus sp. 1XD22-93 and Bacteroides uniformis may be involved in CKD bacterial dysbiosis. Bacteroides uniformis has previously been found to generate beta-lactamase (Bacteroides uniformis play a more critical role in CKD. At the same time, Lactobacillus johnsonii and Lactobacillus intestinalis were shown to be the most important contributing species in healthy colon compared to rats with renal disease. In a recent study, Lactobacillus johnsonii was shown to be able to reduce inflammatory response in human (Lactobacillus johnsonii inhibited the microbial metabolism of tryptophan, preventing tryptophan from being transformed into harmful PBUTs and allowing it to be absorbed directly by the body. It indicated that in CKD, maintaining the homeostasis of Lactobacillus johnsonii may reduce the microbial metabolism of tryptophan, hence decreasing its conversion to toxic metabolites. Thus, Lactobacillus johnsonii deficiency may exacerbate the progress of CKD. Importantly, deficiencies of these beneficial species in the intestinal flora of CKD rats can be reversed by FMT from normal flora. Taken together, enrichment of harmful bacteria and loss of beneficial bacteria are the main flora characteristics of CKD, which are committed to the deterioration of CKD. Nevertheless, FMT from normal flora to renal disease can effectively colonize beneficial bacteria to reduce organism damage.Disturbance of intestinal flora existed in CKD and long-term flora disturbance will promote the deterioration of CKD . The haractamase . And in actamase . Meanwhiactamase . These fin human . ResultsPathogenic metabolome changes caused by alteration in the microbiome have been given a lot of attention in research on renal health . It is aSeveral studies have been dedicated to reducing PBUT levels in CKD. However, only some kinds of toxins in PBUTs could be reduced and it’s hard to get back to normal level . ExcitinThe findings of this experiment imply that FMT can be used to prevent CKD patients from acquiring malignant lesions, particularly without organic disease. Recent research has resulted in the establishment of uniform rules, processes for standardized donor selection, and procedures for standardized microbiological screening for stool preparation . The appIn conclusion, the findings of this study indicate that restoring the intestinal environment with FMT is an effective therapy for CKD. FMT lowers the accumulation of PBUTs in the host by rectifying the gut microbiota amino acid metabolism, hence reducing the progression of CKD. Our findings also imply that inhibition of lysine degradation could be a therapeutic target for the reduction of PBUTs. In general, our data provide crucial evidence for the FMT treatment of CKD patients.PRJNA874518.The original contributions presented in this study are publicly available. This data can be found at: NCBI repository, BioProject accession number: The animal study was reviewed and approved by the Animal Experimentation Ethics Committee of the China Agricultural University.XL, XFW, XYW, and FR: conceptualization. XL, MZ, and YL: methodology. XL, PL, and LW: investigation. XL: formal analyses and writing—original draft. XL and MZ: visualization. MZ: validation. XFW: data curation. XL, MZ, XYW, and FR: writing—review and editing. XYW: project administration. FR: supervision. All authors have read and agreed to the published version of the manuscript.


The PI3K/AKT/mTOR signaling pathway may play crucial roles in the pathogenesis of obesity and diabetes mellitus, as well as metabolic syndromes, which could also be risk factors for cardio-metabolic disorders. Consistently, it has been shown that beneficial effects may be convoyed by the modulation of the PI3K/AKT/mTOR pathway against the development of these diseases. Importantly, the PI3K/AKT/mTOR signaling pathway can be modulated by probiotics. Probiotics have a variety of beneficial properties, with the potential of treating specific diseases such as immune-related diseases, which are valuable to human health. In addition, an increasing body of work in the literature emphasized the contribution of genetically modified probiotics. There now seems to be a turning point in the research of probiotics. A better understanding of the interactions between microbiota, lifestyle, and host factors such as genetics and/or epigenetics might lead to a novel therapeutic approach with probiotics for these diseases. This study might provide a theoretical reference for the development of genetically modified probiotics in health products and/or in functional foods for the treatment of cardio-metabolic disorders. Dyslipidemia has been established as an important factor that is connected with the pathogenesis of several diseases including atherosclerosis, hypertension, cardiovascular disease, obesity, and diabetes mellitus. Therefore, cardio-metabolic disorders are a set of pathophysiological conditions resulting from a disturbance in hepatic lipid metabolism, which may occupy a position at the crossroads of cardiovascular disease and metabolic disorder . The risThe potential therapeutic approaches for the improvement of various diseases might target gut microbiota and/or their metabolites by following the alteration of pathological pathways in diseases . One of Phosphoinositide 3-kinase (PI3K) and AKT, a serine/threonine protein kinase also known as protein kinase B, are key components with the mechanistic target of rapamycin (mTOR) for signaling network , possiblGlucose uptake through GLUT4 might be stimulated by anthocyanin via the upregulation of the PI3K/AKT and adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathways . AMPK isAkkermansia muciniphila (A. muciniphila) administration [Lactiplantibacillus plantarum (L. plantarum) treatment could also enhance the expression of PI3K/AKT in the liver, which could prevent high-fat-diet-induced glucose tolerance and/or hyperglycemia to improve type 2 diabetes mellitus [Lactobacillus sp. [Lactobacillus rhamnosus (L. rhamnosus) could prevent cytokine-induced cellular apoptosis in gut epithelial cells by up-regulating the PI3K/AKT signaling pathway [Bifidobacterium animalis subsp. lactis (B. lactis) strain could retard an apoptosis of the colonic epithelial cells by up-regulating the PI3K/AKT signaling pathway [B. lactis combined with L. plantarum could regulate the growth of malignant glioma by suppressing the PI3K/AKT pathway in mice [Probiotics, as naturally occurring bacteria that are detected in human and/or animal intestine, revealed their capability against the development of various diseases via the alteration of intracellular signaling pathways within target cells. Importantly, the PI3K/AKT signaling pathway could be successfully modulated by probiotics , which mstration . Lactiplmellitus ,41. Altellus sp. . Lactoba pathway . Probiot pathway . Bifidob pathway . Interes in mice .L. plantarum could activate AMPK for reducing the production of ROS and inflammation, which might contribute to inhibiting atherosclerosis [L. plantarum supplementation could adjust the AMPK signaling pathway, which could amend the insulin signaling pathway, fatty acid biosynthesis, fatty acid metabolism, and the glucagon signaling pathway [A. muciniphila could successfully enhance the AMPK activation for the improvement of inflammatory responses and/or the restoration of intestinal barrier function [Streptococcus thermophilus and Lactococcus lactis (L. lactis) could also protect the liver by the activation of AMPK-mediated signaling, thereby promoting lipid oxidation [Bacillus licheniformis could activate AMPK in hepatocytes for the regulation of gene expression associated with lipid metabolism [L. rhamnosus could also activate the AMPK pathway, which may have positive effects on hyperlipidemia by lowering the serum lipid concentration and improving the lipid profile [L. rhamnosus may increase the longevity-related bacteria such as Bifidobacterium, Lactobacillus, and A. muciniphila in the gut, which could also activate the AMPK pathway and contribute to the pathogenesis of age-related diseases [As mentioned above, AMPK is a significant molecule involved in regulating biological energy metabolism  and/or iclerosis . On the clerosis . It is w pathway . A. mucifunction . A blendxidation . Bacillutabolism . L. rham profile . Interesdiseases .Faecalibacterium prausnitzii is one of the most abundant anaerobic bacteria in the gut of healthy individuals that can produce SCFAs such as butyrate. A strain of Lactobacillus paracasei was initially isolated from the feces of an elderly Italian person, which could prevent and/or improve the pathology of type 2 diabetes mellitus by reduction in inflammation via the alteration of the PI3K/AKT pathway and the production of gut-microbiota-derived metabolites such as SCFAs [L. rhamnosus, the concentration of acetic acid, propionic acid, and butyric acid might be significantly raised [L. rhamnosus for acquiring epigenetical effects in probiotics. Obesity is a complex pathology with a multifactorial pathogenesis linked with lifestyle and epigenetic factors [L. rhamnosus supplementation use as a probiotic could contribute to a significant decrease in plasma triglycerides, low density lipoprotein (LDL)-cholesterol, insulin, and homeostatic model assessment for insulin resistance (HOMA-IR) [L. rhamnosus could bring improvement in the profile of total cholesterol, LDL, high density lipoprotein (HDL), triglycerides, and weight gain [L. rhamnosus could activate the AMPK pathway, and reduce the gene expression of peroxisome proliferator-activated receptor (PPAR) [L. rhamnosus could down-regulate the expression of genes related to adipogenesis and/or lipogenesis in high-fat-diet-fed obese mice [Living microorganisms may have a valuable effect, with probiotics being beneficial for gut microbiota in human health . Therefoas SCFAs   . In addight gain . L. rhamr (PPAR) . In addiese mice .Lactobacillus and Bifidobacterium species are important in folic acid production [One of the SCFAs, butyrate, a major end product of the bacterial fermentation of indigestible carbohydrates, might have positive effects on body weight control and insulin sensitivity . Sodium oduction , which moduction . UnderstLactococci and Lactobacilli have been broadly studied and frequently manipulated for the progress of engineered probiotics [L. lactis has been used as a food-grade and endotoxin-free genetically engineered vector for protein expression [L. lactis is a Gram-positive bacterium commonly used in the production of dairy foodstuffs. In addition, this bacterium can synthesize bacteriocins [L. lactis strains expressing murine IL-10 or IL-35 are considerably therapeutic in the dextran sodium sulfate (DSS)-induced colitis in a mouse model [L. lactis expressing IL-35 could also protect against rheumatoid arthritis in mice [L. lactis has improved functional β-cell mass and hyperglycemia without any diabetic symptoms in the non-obese diabetic mouse model [Mycobacterium HSP65, when expressed in L. lactis, could also be protective against DSS-induced colitis [L. lactis engineered to express HSP65 might be an effective therapeutic method in preventing diabetes mellitus type 1 [L. lactis is a glucagon-like protein-1 (GLP-1) receptor agonist that is thought a good therapeutic peptide for type 2 diabetes, which might theoretically serve as a novel strategy for oral treatment of diabetes [Lactococcus-expressing Ling Zhi-8 may be a promising treatment for improving MAFLD [Lactococci and Lactobacilli strains have also been engineered to address the induction of superoxide dismutase expression for reducing reactive oxygen species (ROS) and/or various oxidative stresses [Lactobacillus casei could diminish retinopathy symptoms in diabetic retinopathy mouse models [obiotics . In partpression . L. lacteriocins , which ceriocins . For exase model ,79. L. l in mice . It is w in mice  (Figure se model . Heat sh colitis . In addis type 1 . Exendindiabetes . Exendinng MAFLD . Ling Zhng MAFLD ,87. It hng MAFLD ,87. Probstresses . Human ae models ,90. It ie models . Recombie models .In these ways, engineered probiotics are now demonstrating an effective delivery method that could prevent inflammatory diseases ,93. The L. lactis has been further defined [L. lactis from several weeks to 3 days [Lactobacilli are being developed as targeted therapies against a wide range of diseases such as IBD and viral/bacterial infections [Lactobacillus reuteri [Lactobacillus plantarum, CRISPR/Cas9-mediated genome editing has been also presented [Bacillus subtilis [The mechanism of probiotics could be, in part, concluded as an alteration or recovery of microbiota balance. Helpfully, engineered probiotics could serve as optimum vectors to produce beneficial molecules targeting specific factors, cells, organs, and/or even pathogens . The dev defined . In combo 3 days . Engineefections . In addi reuteri . CRISPR  reuteri . In Lactresented . Furthersubtilis . In thessubtilis  for the subtilis . Meanwhi


Sparganium angustifolium was sequenced and annotated in the present study. The circular genome is 161,720 bp in length and exhibits a typical quadripartite structure with a large single-copy  and small single-copy  regions, separated by a pair of inverted repeats . The cp genome contains 114 unique genes, including 80 protein-coding, 30 tRNA, and four rRNA genes. The phylogenetic analysis of Typhaceae strongly supported the monophyly of Sparganium and resolved two clades that represented newly revised two subgenera. S. angustifolia has the closest relationship with S. emersum in the present sampling extent.The complete chloroplast (cp) genome of  Sparganium (Typhaceae) is a marsh or aquatic genus including approximately 14 species distributed mainly in temperate and cool regions  different from its congeners  C.D.K.Cook & M.S.Nicholls 1867, S. stoloniferum (Buch.-Ham. ex Graebn.) Buch.-Ham. ex Juz. 1934, S. fallax Graebn. 1900, S. glomeratum (Laest. ex Beurl.) Beurl. 1853 and S. stoloniferum subsp. choui (D.Yu) K.Sun 1992 have been reported  in Jilin Province of China, and dried with silica gel. The specimen was deposited in Herbarium of the Wuhan University  under the voucher number Xu3837. The plant material does not involve ethical conflicts. All the collection and following sequencing work was strictly executed under local legislation and related laboratory regulations to protect wild resources. Total genomic DNA was extracted using the DNA Plantzol Reagent  following the manufacturer’s protocol. Library preparation and genomic DNA sequencing on the BGISEQ-500 platform were conducted by the Beijing Genomics Institute . The obtained paired-end reads were used to assemble the cp genomes using NOVOPlasty4.2  gene from S. stoloniferum (GenBank accession no. NC_044634) as the seed. The cp genome annotation was performed in program Geneious Prime v2020.0.5  separated by a large single-copy  region and a small single-copy  region. The overall GC content is 36.9%. The IR regions have a higher GC content (42.4%) than the LSC (34.8%) and SSC (31.0%). The cp genome encodes a set of 114 unique genes, including 80 protein-coding, 30 tRNA, and four rRNA genes. Among the genes identified, nine genes  contain one intron and three genes  comprise of two introns. Twenty genes  are duplicated in the IR region, and ycf1 is pseudogene.The cp genome of Ananas comosus (L.) Merr. 1917 and Tillandsia usneoides (L.) L. 1762 were set as outgroups. The phylogenetic analysis provided strong support for monophyly of Sparganium (100% BS) and two main clades were recovered, which represented two Sparganium subgenera proposed by Sulman et al. (S. angustifolia has the closest relationship with S. emersum (Chloroplast genomes of 15 Typhaceae species and two Bromeliaceae species were downloaded from GenBank for the phylogenetic analysis. A total of 18 sequences were aligned using the default settings in MAFFT v7.3 (Katoh and Standley n et al. . In the  emersum .QL and ZY conceived the idea; QL contributed to the sampling; XC, JL, and JY collected the data; WM analyzed the data. The manuscript was written by ZY and WM. All authors read and approved the final manuscript.


Aquatica leii and terrestrial Lychnuris praetexta), and then a set of metabolites and their pathways involved in freshwater adaptation of aquatic firefly species were investigated by intraspecific and interspecific metabolomics comparisons, as well as by functional enrichment analysis. These molecules and pathways were primarily involved in oxidative stress/xenobiotics/immunity response, energy metabolism, sense function, and morphological attributes related to the freshwater lifestyle of aquatic A. leii. This study suggests that abundance-level changes in metabolites contributed to freshwater adaptation of fireflies, and provides insights into the metabolic mechanisms of aquatic adaptation in insects.To date, little is known about the molecular mechanisms of aquatic adaptation in invertebrates, particularly in insects, which are the most abundant and diverse species. How aquatic insects adapt to freshwater environments remains largely unknown. Fireflies have terrestrial and aquatic lineages according to the habits of their larvae, and provides a good opportunity to explore aquatic adaptation of insects. We generated adult and larval metabolomes of two firefly species (aquatic Aquatica leii (freshwater) and Lychnuris praetexta  to identify freshwater adaptation-related metabolites (AARMs). We identified 110 differentially abundant metabolites (DAMs) in A. leii (adults vs. aquatic larvae) and 183 DAMs in L. praetexta . Furthermore, 100 DAMs specific to aquatic A. leii larvae were screened as AARMs via interspecific comparisons (A. leii vs. L. praetexta), which were primarily involved in antioxidant activity, immune response, energy production and metabolism, and chitin biosynthesis. They were assigned to six categories/superclasses . Finally, ten metabolic pathways shared between KEGG terms specific to aquatic fireflies and enriched by AARMs were screened as aquatic adaptation-related pathways (AARPs). These AARPs were primarily involved in energy metabolism, xenobiotic biodegradation, protection of oxidative/immune damage, oxidative stress response, and sense function , and certain aspects of morphology . These results provide evidence suggesting that abundance changes in metabolomes contribute to freshwater adaptation of fireflies. The metabolites identified here may be vital targets for future work to determine the mechanism of freshwater adaptation in insects.Aquatic insects are well-adapted to freshwater environments, but metabolic mechanisms of such adaptations, particularly to primary environmental factors , are poorly known. Most firefly species (Coleoptera: Lampyridae) are terrestrial, but the larvae of a few species are aquatic. We generated 24 global metabolomic profiles of larvae and adults of  Compared with terrestrial habitats, fresh water possesses a relatively lower content of oxygen (hereafter referred to as hypoxia) and light, water pressure, as well as more abundant pathogenic bacteria communities ,2. ThesePlectropomus leopardus) showed greater melanin synthesis activity in black compared to red-colored groups [Colossoma macropomum) faced hypoxia, anaerobic metabolism rapidly adjusted glucose and lactate metabolism, providing adequate energy [Despite the accumulation of metabolite information and the wide use of mass-spectrometric techniques in metabolomics, only a few studies have explored the metabolic mechanisms underlying aquatic adaptations of animals. For example, body color is an important ecological trait in aquatic animals, influencing their survival ,12. Metad groups . When tae energy . OverallAedes mosquitoes [Aquatic insects (defined by having at least one aquatic life stage) make up 80% of total aquatic animal diversity ,14. Mostsquitoes . This imAsymmetricata, Lychnuris and Pteroptyx), aquatic (Luciola and Aquatica), and semi-aquatic lineages (Pygoluciola) [The Lampyridae family (Insecta: Coleoptera: Cantharoidae), commonly known as fireflies, includes at least 2000 recorded species belonging to 100 genera, widely distributed and found in tropical, subtropical, and temperate zones ,18. Theyluciola) . Among tluciola) ,20. In rluciola) . Genome-Aquatica leii and terrestrial Lychnuris praetexta). Then, metabolites with significant changes in A. leii were identified by comparing differentially abundant metabolites (DAMs) between both species. Finally, functional analyses  were performed on candidate metabolites involved in freshwater adaptations.Here, we sequenced and intraspecifially and interspecifically compared the adult and larval metabolic profiles of two firefly species (aquatic A. leii and L. praetexta were raised as previously described (A. leii freshwater larvae (ALL) and terrestrial adults (ALA) were randomly collected, to a total of 50 4th instar, 50 5th instar, and 30 6th instar larvae, and 20 female and 20 male adults. We additionally randomly collected larvae (LPL) and adults (LPA) of L. praetexta inhabiting terrestrial environments across the entire life cycle, to a total of 10 4th instar, 6 5th instar, and 5 6th instar larvae, and 20 female and 20 male adults. Each collected sample per developmental stage was put in a 10 mL plastic centrifuge tube and then stored at −80 °C. The sampling was independently repeated six times as six biological replicates. The intestines were removed from all individuals to avoid disturbance of residual foods and gut microbes. A flow diagram of the experiments and data analyses is shown in Larvae (4th–6th instars) and adults of g at 4 °C for 15 min , the supernatant of each developmental stage of 4th–6th instars of A. leii samples was equally mixed as ALL samples and used for further global metabolomics analysis. Adult A. leii, 4th–6th instars of L. praetexta, and adult L. praetexta samples were also similarly processed as ALA, LPL, and LPA samples, respectively. In total, the samples were used to generate 24 metabolic profiles .For metabolite extraction of each replicate, 50 mg of firefly tissue at each developmental stage was mixed with 400 μL of pre-cooled extracting solution  in a 2 mL Eppendorf tube, and then a 6 mm diameter grinding bead was added. After grinding for 6 min at 50 Hz at 10 °C in a Wonbio-96c high-throughput tissue crusher , the samples were extracted at 4 °C at 40 kHz for 30 min using a controlled-temperature ultrasonic cleaning machine . The samples were placed at −20 °C for 10 min for proteins to precipitate. After centrifugation at 30,000× n = 3). The QC samples were sequenced parallel to the experimental samples, which represented the whole sample set, and were injected at regular intervals to monitor stability in the sequencing process.In addition, to avoid systematical and technical errors caused by metabolome sequencing, 5 µL from each collected supernatant sample was mixed into a 2 mL Eppendorf tube as a quality control sample  mode, controlled by the acquisition software . In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The electrospray ionization (ESI) source conditions were set as follows: sheath gas flow rate of 50 arb, aux gas flow rate of 13 arb, capillary temperature 325 °C, normalized collision energy 204,060 eV, full MS resolution at 60,000, resolution (MS/MS) at 7500, collision energy at 10/30/60 in normalized collisional energy (NCE) mode, and spray voltage at (+) 3.5 kV or (–) 3.5 kV. Raw data of the 24 metabolic profiles of ALL, ALA, LPL, and LPA (n = 6) were obtained by collecting data in positive and negative ion modes.Global metabolomics analysis was implemented using liquid chromatography with tandem mass spectrometry (LS-MS/MS) as previously described , using cm/z) values, and peak intensity. Metabolic features that were detected at least in 70% of samples were retained. After filtering, minimum metabolite values were assessed for samples in which the metabolite levels fell below the lower limit of quantitation. Peak annotation was carried out using Compound Discover v3.0 (Thermo Fisher Scientific) and OSI-SMMS v1.0 , integrated with the mzcloud database . An internal standard was used for data QC (reproducibility). Metabolic features with relative standard deviation (RSD) of QC > 30% were discarded. Following normalization procedures and imputation, statistical analysis was performed on log-transformed data to identify significant differences in metabolite levels between comparable groups. Subsequently, the acquired MS/MS spectra were matched against metabolome databases including HMDB , KEGG , and an in-house MS/MS database . The MS/MS spectra match score was calculated using a dot-product algorithm and ranged from 0 to 1. The cutoff for match score was set as 0.4. The data were normalized by the internal standards before statistical analysis. The Pearson correlation coefficient was calculated between each sample pair to evaluate the repeatability of the biological replicates. Principal Component Analysis (PCA) was performed to separate and classify the sample groups based on metabolic profiles using SIMCA-P+ software v16 .The raw data were converted into the mzXML format by using ProteoWizard, and processed using R package XCMS (version 3.2) for peak detection, extraction, alignment, and integration, as previously described ,22. The p-values were further corrected by the Benjamini-Hochberg adjustment method , |log2FC(fold change)| > 1, and VIP (the variable importance of projection) > 2. The metabolites involved in freshwater adaptation of larval A. leii were detected as previously described . Briefly, the DAMs of L. praetexta were intersected with that of A. leii, thus excluding non-responding metabolites to fresh water in larval A. leii. Subsequently, DAMs that were only detected in A. leii (A. leii-specific DAMs) were retained as metabolites that presented abundance changes to freshwater environments; these metabolites were identified as aquatic adaptation-related metabolites (AARMs) in A. leii larvae.The significance of each DAM between larvae and adults of each firefly species was evaluated using unpaired Wilcoxon tests. The http://www.metaboanalyst.ca/, accessed on 3 May 2022). Benjamini-Hochberg adjustment was employed as the FDR correction method. To detect reliable KEGG pathways specifically involved in freshwater adaptation of larval A. leii, two methods  were used, as previously described . In the DAM-KEGG-intersection method DAMs of L. praetexta and A. leii were subjected to KEGG enrichment analysis, their results were intersected, and the pathways specific to A. leii were obtained. In the AARM-KEGG method, the AARMs above obtained were directly used for KEGG enrichment. The results shared by both methods were considered as the final KEGG metabolic pathways associated with freshwater adaptation of A. leii .According to the HMDB database annotation information, AARMs were divided and assigned to three levels: superclass, class, and subclass. In addition, the identified differential metabolites were further analyzed to determine the relevant biological pathways using MetaboAnalyst 5.0  and negative (ESI−) ion mode, respectively) were detected by the mass spectrometer. Through further screening, 20,276 peaks  were obtained. By further screening peaks, a total of 2928 metabolites (2030 metabolites in ESI+ and 938 metabolites in ESI−) were identified. The correlation value was close to 1 in the ESI+ A and ESIIn total, 100 AARMs were identified . AccordiA. leii larvae. Within the six superclasses, organic acids and derivatives contained the second largest number (12) of AARMs. This superclass included carboxylic acids and derivatives , keto acids and derivatives, and peptidomimetics  were significantly enriched to 14 KEGG pathways  were significantly enriched to eight KEGG pathways  detected in this study possess antioxidant activity [Katsuwonus pelamis) dark muscle [Strigomonas culicis [Larimichthys crocea) [A. leii larvae to hypoxia in fresh water. Antioxidants protect animal cells against DNA and cell damage, cytotoxicity, and apoptosis under hypoxia  by reveractivity , such asactivity  and isolk muscle . In addik muscle . Cynarosk muscle . Treatmek muscle . The inc culicis  and in l crocea) . This inCyprinus carpio) [Eogammarus posjeticus significantly changed under various water pressures [Odontesthes bonariensis, Oncorhynchus mykiss, Oryzias melastigma) were exposed to organic pollutants, the metabolism of 6-succinoaminopurine significantly changed to enhance immune defense [In addition, the animal immune response is highly sensitive to abiotic and biotic stresses such as hypoxia, water pressure, and water pollutants/microbes. For example, tissue oxygen deficit caused by the exogenous pollutant fipronil, an important commonly used insecticide, could induce nonspecific immunity in common carp ( carpio) , and theressures . When fi defense . Canarig defense . In the  defense . Previou defense ,68. In p defense , while t defense . Bisnorc defense . In the  defense .Cuticle chitin biosynthesis (including the chitin-containing exoskeleton) contributes to an adaptation to the increasing water pressure and both the size and morphology of the open tracheal system  in insects . In thisA. leii larvae compared with adults. Previous studies reported a greater amount of lipids and lipid-like molecules in aquatic than in terrestrial insects [A. leii larvae to water pressure.More than half of AARMs were assigned to various categories at different levels, according to the HMDB database. Lipids and lipid-like molecules included the largest number of AARMs, and most of them were up-regulated in aquatic  insects . In gene insects , and ca  insects . AbundanA. leii. Organic acids are intermediate products of metabolism, and play important roles in various cellular biochemical pathways, such as energy metabolism and detoxification [Callosobruchus chinensis larvae increased when exposed to hypoxia [A. leii in fresh water. In addition, nucleosides, nucleotides, and analogues are a diverse group of endogenous metabolites widely present in organisms [Anopheles sinensis larvae under deltamethrin exposure [Interestingly, steroids and steroid derivatives belonging to lipids and lipid-like molecules have been widely found in aquatic crustaceans, some of which act as hormones used to regulate the molting of arthropods . AARMs ification . A study hypoxia . In addi hypoxia ,76,77. Trganisms . DNA canrganisms . In the exposure , and orgexposure . AARMs cCallosobruchus chinensis larvae when exposed to hypoxia [A. leii larvae in freshwater environments by promoting riboflavin-mediated energy metabolism. In addition, purine metabolism is an important pathway in mammalian oxidative stress adaptation [Tursiops truncatus) during diving [A. leii larvae [Metabolites belonging to the glycine, serine, and threonine metabolism pathway significantly changed in  hypoxia . Togethe hypoxia , the gly hypoxia , also co hypoxia . Moreove hypoxia . The antaptation . For exag diving . In the g diving . Biotin i larvae , suggestA. leii in the present study. This evidence shows the importance of metabolic pathways involved in the olfaction and taste functions for freshwater adaptation of insects. In addition, as a key pathway that impacted the timing of molting and metamorphosis of insects [A. leii. This reflects how the aquatic lifestyle of insects may benefit from adaptive changes in timing of molting and metamorphosis.Several studies have shown differences in olfaction triggered by chemosensory-related genes  between terrestrial Lepidoptera and aquatic Trichoptera insects . Indeed, insects , the steThis study explored the metabolic mechanisms regarding adaptation of insects to freshwater environments using global metabolomic profiling of the Lampyridae family. We documented candidate adaptive changes in the metabolites involved in antioxidant activity, immunity, energy metabolism, and morphological attributes  related to the freshwater lifestyle. These metabolites were assigned to categories linked to responses to freshwater environmental stressors . Furthermore, we also identified metabolic pathways that likely contribute to firefly freshwater adaptation. The single species used for freshwater and terrestrial fireflies in this study likely limited the applicability of information. Thus, to expand the base of evidence supporting the metabolite candidates obtained here, it is important to sequence metabolic profiling of more firefly species in the future.


Daphnia longispina complex (Crustacea: Cladocera) contains several keystone freshwater species such as D. longispina O.F. Müller (D. rosea Sars is a junior synonym), D. galeata Sars, D. cucullata Sars, and D. dentifera Forbes. The complex is common throughout the Holarctic, but there are several geographic regions where local forms have been assigned to European species names based on a superficial morphological resemblance. Here we examine the species status of a form that was previously assigned to D. rosea from a montane bog pond on Honshu, Japan. We used two nuclear non-coding loci (nDNA), mitochondrial sequences (the ND2 protein-coding region) and morphology for evidence. The mitochondrial gene evidence supported the existence of a divergent lineage that is more closely related to D. galeata than to D. dentifera. However, morphology and the nuclear DNA data indicated a lineage that is most closely related to D. dentifera. As our evidence supported the existence of a cohesive divergent lineage, we described a new species, Daphnia japonica sp. nov. Recognition of local and subalpine diversity in this group is critical as ongoing anthropogenic disturbance has been associated with introductions, local extirpations, and hybridization.The  Daphnia O.F. Müller (Crustacea: Cladocera) are model organisms in aquatic toxicology, ecology, and evolutionary genetics  Colbourne, Wilson & Hebert; D. (Ctenodaphnia) Dybowski & Grochowski and Daphnia s. str. DNA sequence evidence  supported these divergent subgenera , D. galeata Sars, D. cucullata Sars, and D. dentifera Forbes. A number of factors has complicated the taxonomy: hybridization, introgression, phenotypic plasticity, and introductions  have agreed with nuclear and morphological evidence (such as Daphnia umbra), several divergent 12S rRNA gene sequence lineages  have lacked support from nuclear DNA markers and morphological evidence. Daphnia longispina complex lacked nuclear genetic divergence from three common species. They proposed that ancient mitochondrial introgression may have contributed to the unusual divergence with 12S rRNA genes compared to nuclear markers and morphology. PCR amplification of nuclear copies of mitochondrial genes may also contribute to mitonuclear discordances. Assays for pseudogenes (intact ORF’s and tests of purifying selection) are more straightforward for protein coding genes (compared to 12S rRNA gene sequences). A lack of knowledge of evolution can also affect the taxonomic use of DNA-based markers. Mitochondrial rRNA gene studies revealed several local divergent lineages within the na group . While sDaphnia japonica sp. nov.Here, we assess the species hypothesis for a montane form that we detected in Misumi-ike, Japan, which has yet to be examined at a genetic level. We use nuclear DNA sequences, morphology, and protein-coding mitochondrial gene sequences and link this form with additional montane populations and formally describe a new endemic species, http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:7AAA1961-81B8-46D8-8D0A-9FE1F5CD776D. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central SCIE and CLOCKSS.The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix Plankton samples from Japan were collected by Dr. S. Ishida during his Ph.D. dissertation work, such sampling does not require special permission in Japan. The species was not assessed as endangered at the time of collection and is currently not subject to specific regulations, however all efforts were taken to ensure that the collection and preservation of animals was performed with due consideration of their welfare. The number of individuals taken did not represent a significant proportion of the clonal populations present at each site.Daphnia longispina group  and one ND2 sequence from an unnamed lineage (Daphnia longispina complex using Geneious (https://www.geneious.com). Sequences (and their genomic locations) were identified from the reference genome of Daphnia galeata (Daphnia dentifera (4 mg dry mass). This total DNA was extracted using Nucleospin gDNA Cleanup (Macherey-Nagel) and exposed to restriction enzyme digestion with Rsa I (New England Biolabs). Purified DNA was size selected (1–3 kbp) using a 2% agarose gel, and further purified using Nucleospin gel Cleanup. After dephosphorylation using Shrimp Alkaline Phosphatase, we performed cloning with a Zero Blunt TOPO PCR cloning kit . Colonies were then sequenced and primers designed. Of these, two nuclear regions were successfully amplified and sequenced from the target specimens of Misumi-ike. We used the following PCR primers (custom oligos from Integrated DNA Technologies):Ethanol-preserved specimens of the  lineage  in the D galeata  by BLASTLocus 1 ,Daphnia galeata genome indicated that these loci are present as intergenic single copies and located on contig CAKKLH010000168 from bases 1032297 to 1032629 (locus 1) and contig CAKKLH010000342 from bases 156848 to 157308 (locus 2). Cloning  was used for sequences from putative hybrids.Locus 2 . A nucleotide BLAST to the reference https://www.geneious.com) with the species identification plugin.The ND2 region was aligned by translation alignment in Seaview  using thDaphnia dentifera  in a drop of a glycerol-ethanol mixture. Then, 10 parthenogenetic females, five ephippial females, two juvenile and three adult males were dissected under a stereoscopic microscope using tungsten needles . The test of relaxed selection for the mitochondrial branch leading to D. japonica was not significant . However, one published ND2 sequence  had the longest branch on the amino acid tree (p = 0.001).The alignment lengths were 930 nt for the ND2 gene, 480 nt for nuclear locus 1, and 501 nt for nuclear locus 2. The mitochondrial locus  supportelineages . Howevercid tree  and signD. japonica sp. nov. with specimens from populations proposed by D. japonica sp. nov. within the D. galeata/D.cucullata clade using both nucleotide  from D. galeata for ND2 and 0.12 (K2P+G4) from D. dentifera for nuclear locus one. For nuclear locus one, the divergent lineages from Honshu  O.F. Müller, 1785Subgenus Daphnia ( Daphnia) longispina species complexDaphnia japonica sp. nov.7Daphnia rosea Sars in Daphnia rosea Sars, 1862: 268–269).“New species lineage” in Etymology. The species is named after the country of the type locality—“of Japan” in Latin.Type locality. A small, shallow bog-lake Misumi-Ike , Yamagata Prefecture, N part of Honshu Island, Japan. The type series was collected in October 2006 by S. Ishida.Holotype. A parthenogenetic female, MGU Ml-253 in the Collection of Zoological Museum of Moscow State University, Moscow, Russia.Allotype. Adult male, MGU ML-254.Additional material examined from the type locality. Fifty juvenile males and females, adult parthenogenetic, ephippial females and males, MGU Ml-255. Many juvenile males and females, adult parthenogenetic, ephippial females and males, AAK M-300 in the personal collection of A.A. Kotov, A.N. Severtsov Institute of Ecology and Evolution, Moscow, Russia.Short diagnosis. Parthenogenetic female with very large head, low anterior crest (but without traces of helmet) and long, straight rostrum; posterior margin of head flat. Abdomen with first abdominal process almost straight, directed anteriorly, second process shorter, third process as a massive mound on the segment, fourth process absent. Antenna I body as minute hillock. Length of stiff seta on inner-distal portion of limb II (=rigid seta) 2/3 length of soft seta. Juvenile females and males with few neckteeth. Dorsal wall of ephippium forming dorsal plate without spinules. Adult male head with rounded rostrum, massive projection posterior to antenna I base. Distal most head extremity projected ventrally, fully occupied by large compound eye. Abdomen with all abdominal processes reduced, small mound present on each first, second and third segments. Gonopore opens subdistally, without genital papilla. Antenna I (antennule) long, almost straight; antennular setae small, its tip projected beyond antennular body tip. Male seta (flagellum) located on low process, longer than aesthetascs, bisegmented, with hooked tip. Length of females 0.9–2.1 mm, adult males 1.3–1.6 mm.Adult parthenogenetic female. Body subovoid in lateral view, maximum height in body middle  present in male , ephippial females 1.7–2.0 mm (n = 5), juvenile males 0.7–0.8 mm (n = 10), adult males 1.3–1.6 mm (n = 5).Differential diagnosis.D. japonica sp. nov. differs from the species of the D. longispina species complex known from Japan in a series of characters  and Lake Imori-ike , Niigata  . The genetic and morphological evidence supports the existence of a novel species of Daphnia longispina complex is a geographically widespread syngameon  introgressed lineages in weakly glaciated regions such as Japan. We have no calibrated molecular clock for among-population divergences that includes Misumi-ike specimens. If the lineages of D. japonica, then the divergences for mtDNA among populations suggest that the species origin predates the last glaciation. Indeed, D. dentifera/D. longispina about 15 MYA.As the yngameon , introgryngameon . Recent mendotae  and someex group . Ishida D. japonica sp. nov. can coexist with extant members of the complex. However, members of the D. longispina complex are almost never detected in very low pH waters  than is known for D. galeata. Non-plastic morphological differences among species of the Daphnia longispina complex  are few in number. However, the abdominal processes of parthenogenetic females and males appear to have diagnostic value in the group  are usually phenotypically plastic, but plasticity need not preclude taxonomic informativeness. For example, the reaction norms of helmet shape for the entifera . Likewisthe head , but sucthe head . We have galeata , D. japohe group .D. japonica sp. nov. as a divergent lineage related to D. galeata/D. cucullata , there may be an undetected species in this group that donated this genome to D. japonica sp. nov. The results support the contention that the analyses of both nuclear and mitochondrial markers are necessary when divergent mitochondrial haplotypes are detected in this species complex (D. japonica sp. nov. is an endemic taxon known only from Honshu.As the mitochondrial gene trees group  complex . PresentDaphnia longispina complex (longispina-like morphology must be carefully assessed for cryptic species. Some of these are likely undescribed species, such as the Daphnia from Berse in Norway (Our findings follow a series of discoveries of montane and subarctic lineages of the  complex . Populatn Norway . UnderstDaphnia that is likely present in but a few mountain lakes and ponds in Japan. The daphniid fauna of many Japanese and other eastern Palearctic waters have recently been studied in detail with genetic markers (Daphnia umbra and Daphnia lacustris are presently unknown from the eastern Palearctic (Here we describe a new species of  markers . Also no markers . Palearclearctic .D. japonica sp. nov. appears to represent such a pre-Pleistocene relict in agreement with Daphnia from East Asia. We describe Daphnia japonica sp. nov. from a montane bog on Honshu Island. The mitochondrial phylogeny was discordant with the morphological and nDNA similarity to D. dentifera. The finding continues a trend of hidden diversity in ecologically sensitive montane habitats and in East Asia for Daphnia.We find genetic and morphological evidence that supports the existence of a new endemic species of 10.7717/peerj.14113/supp-1Supplemental Information 1Locations for the NCBI sequences of the ND2 mitochondrial gene region used in the present study.Click here for additional data file.10.7717/peerj.14113/supp-2Supplemental Information 2Anonymous nuclear local alignments and DNA sequences from mitochondrial ND2 for Daphnia japonica in FASTA format.Click here for additional data file.


Listeria monocytogenes is an important foodborne zoonotic bacterium. It is a heterogeneous species that can be classified into lineages, serogroups, clonal complexes, and sequence types. Only scarce information exists on the properties of L. monocytogenes from game and game meat. We characterised 75 L. monocytogenes isolates from various game sources found in Finland between 2012 and 2020. The genetic diversity, presence of virulence and antimicrobial genes were studied with whole genome sequencing. Most (89%) of the isolates belonged to phylogenetic lineage (Lin) II and serogroup (SG) IIa. SGs IVb (8%) and IIb (3%) of Lin I were sporadically identified. In total, 18 clonal complexes and 21 sequence types (STs) were obtained. The most frequent STs were ST451 (21%), ST585 (12%) and ST37 (11%) found in different sample types between 2012 and 2020. We observed 10 clusters, formed by closely related isolates with 0–10 allelic differences. Most (79%) of the virulence genes were found in all of the L. monocytogenes isolates. Only fosX and lin were found out of 46 antimicrobial resistance genes. Our results demonstrate that potentially virulent and antimicrobial-sensitive L. monocytogenes isolates associated with human listeriosis are commonly found in hunted game and game meat in Finland. Listeria monocytogenes has emerged over recent decades as an important foodborne pathogen responsible for numerous outbreaks [L. monocytogenes is responsible for listeriosis, a disease affecting both humans and animals. Foodborne listeriosis typically causes a self-limited gastroenteritis among healthy people [utbreaks . L. monoy people . Howevery people . Invasivy people . Invasivy people .L. monocytogenes is a ubiquitous bacterium that can survive in a variety of environments and grow at low temperatures, e.g., in cold-stored foods [L. monocytogenes, and mammals and birds can spread this pathogen through faecal shedding [L. monocytogenes-contaminated food is an important source attributed to human infections [ed foods . Soil anshedding . L. monofections . The confections ,7.L. monocytogenes has sporadically been found in game animals and on game carcasses [L. monocytogenes in deer and wild boar faeces have been low, varying between 0 and 6% [L. monocytogenes was detected in 44% and 41% of deer and wild boar tonsils, respectively [L. monocytogenes was detected in 5% of deer carcasses in Austria using an antigen test [L. monocytogenes on deer carcasses shows the importance of observing hygiene practices during hunting and slaughtering.arcasses ,9,10,11.0 and 6% . However0 and 6% ,13,14,15ectively . Recentlgen test  and in 1gen test . This reL. monocytogenes is a very heterogeneous species, which can be divided into at least 14 serotypes based on variation in the somatic (O) and flagellar (H) antigens [L. monocytogenes can be divided into four phylogenetic lineages (Lin), six serogroups (SGs), multiple clonal complexes (CCs), and sequence types (STs) [antigens ,19. Overes (STs) ,5. Most es (STs) , whereases (STs) ,22.L. monocytogenes isolates [L. monocytogenes isolates from game and game meat will provide valuable information on the significance of game in the meat production chain and in human infections.Whole genome sequencing (WGS)—an accurate method with a high resolution—is currently becoming the method of choice for characterising isolates . It has isolates . InvestiL. monocytogenes isolates from game sources remain scarce. Game and game meat may play an important role in the L. monocytogenes infection cycle. The aim of our study was to use WGS to investigate the diversity and genetic relationships between L. monocytogenes isolates from hunted game and game meat in Finland. Furthermore, we studied the presence of important virulence and resistance genes obtained from the WGS data. Studies on the genetic relationships between L. monocytogenes has been detected in hunted game and game meat in Finland between 2012 and 2020, especially in deer and mallard meat  according to the manufacturer’s protocol. DNA quality was measured with a NanoDrop™ spectrophotometer  and DNA quantity with a Qubit fluorometer (ThermoFisher Scientific). WGS was performed on the Illumina platform by CeGaT . Illumina DNA Prep library preparation kit and NovaSeq6000 were used to generate 100 bp paired end reads. The short raw reads were assembled de novo using a Unicycler v0.4.8 assembler available on the PATRIC 3.6.12 platform . DNA of http://www.genomicepidemiology.org/services/, accessed on 11 November 2022). In silico typing using 7-gene multi-locus sequence typing (MLST) [https://bigsdb.pasteur.fr/listeria/, accessed on 11 November 2022) platforms. STs obtained through the 7-gene MLST were grouped into CCs and phylogenetic Lin [Species identification was confirmed in silico from the assemblies with KmerFinder v3.2 and SpeciesFinder v2.0  availablg (MLST)  was perfetic Lin .L. monocytogenes isolates were genotyped with core genome MLST (cgMLST) based on 1748 genes [http://www.mgc.ac.cn/VFs/, accessed on 11 November 2022). In total, the presence of 33 virulence genes and 46 AMR genes among the 55 L. monocytogenes isolates was recorded.Assembled sequence data of 55 48 genes  using th48 genes  and the 48 genes  (http://L. monocytogenes isolates from 75 hunted game and game meat samples—isolated in Finland between 2012 and 2020—were serotyped and characterised by seven-gene MLST (L. monocytogenes 4b was found on deer carcasses (n = 3), wild boar organs (n = 2) and in pheasant faeces (n = 1)  . L. monoL. monocytogenes isolates from hunted game and game meat samples (n = 75) were classified into Lin I and II, 18 CCs and 21 STs  and mallard meat (13/75)  . These sL. monocytogenes isolates were characterised with cgMLST based on 1748 genes. In total, 35 CTs among 21 STs were obtained using the BIGSdb-Lm platform  formed by closely related genotypes were obtained with the cgMLST based on 1701 genes using the Ridom software  of the genes were detected in all isolates. Seven virulence genes  were not present in all isolates. We designed 12 virulence profiles (VPs) based on these genes  had the same VP  but serotype 4b  was also found. L. monocytogenes strains belonging to SG IIa/Lin II and SG IVb/Lin I are responsible for the largest share of listeriosis [L. monocytogenes IIa and IVb were found in deer and wild boar tonsils in Spain [L. monocytogenes isolates found in epidemiological studies, surveys and during monitoring.teriosis ,35,36. Hteriosis ,21. Recein Spain . SerotypL. monocytogenes isolates from game origin [L. monocytogenes isolates studied. This was expected because L. monocytogenes isolates were found from various sources and locations during a ten-year period [https://thl.fi/en/web/infectious-diseases-and-vaccinations accessed on 11 November 2022). ST451 (21%) was the most frequently found ST in our study, as it was found in different sample types between 2012 and 2020. This type has also been reported in human listeriosis in Finland yearly between 2017 and 2021. ST451 is a common universal ST found in humans, animals, foods, and the environment in Europe [Very little is known about the genetic diversity of e origin . We founr period ,24,25. Ar period ,31. Mostr period ,31,32. Sn Europe ,31,32,37L. monocytogenes isolates of different STs. This is very understandable because the isolates originated from wild boars hunted in various geographical locations in Finland [L. monocytogenes SG IIa isolates from mallard faeces. The hunted mallards were reared and fed in a natural pond before being hunted, which could be a common contamination source for the mallards [L. monocytogenes is relatively commonly found in various environments, and L. monocytogenes-contaminated soil and water are therefore important L. monocytogenes sources [Wild boar organs were contaminated with several  Finland . Fewer Smallards . L. mono sources ,38. ST18 sources  and Latv sources .L. monocytogenes isolates with 0 to 10 allelic differences among the hunted game and game meat isolates using cgMLST, which is the method capable of differentiating related strains from unrelated ones [L. monocytogenes isolates—forming CL18—were isolated from mallard meat originated from various mallards sampled on the same day in the same game meat processing plant, indicating a cross-contamination during processing. In CL412, four very closely related isolates from mallards were sampled on two different days in the same plant, indicating a plant contamination possibly due to inadequate cleaning. Deer meat isolates also formed two clusters—CL155 and CL451c—both with three very closely related isolates. The isolates in CL155 and CL451c were from deer meat samples cut on different days in the same plant. Cross-contamination during meat cutting occurs easily if working hygiene is poor. L. monocytogenes can easily persist in the plant, and thorough cleaning of the meat processing plant after each working day is therefore very important. We identified some CLs of ted ones . Very clL. monocytogenes has shown heterogeneity in its virulence [L. monocytogenes to spread optimally within the environment [L. monocytogenes is encoded by a wide range of virulence genes [L. monocytogenes isolates of game origin in Finland. The actA gene located on the Listeria pathogenicity island (LIPI-1) was missing in only one isolate (a deer carcass isolate). LIP-I is composed of important virulence genes  and is necessary for intracellular survival and spread from cell to cell [L. monocytogenes strains [actA-negative deer carcass isolate  also missed the lapB (coding for an adhesion protein) and vip (coding for an invasion protein) genes, indicating a reduced virulence in this isolate. The most frequently missing virulence gene ami, which is coding an autolysin protein for adherence, was not found in 42% of the isolates. However, the meaning of ami in the virulence remains unclear. The invasion gene aut was missing only in the isolates belonging to SG IVb. All SG IVb isolates  were aut-negative. The three SG IVb isolates belonging to ST1 were also inlJ-negative. The aut gene codes for an autolysin protein needed for invasion and the inlJ for an internalin protein needed for adherence [L. monocytogenes IVb isolates needs to be further studied. Typically, ST1 (CC1) and ST4 (CC4) have been associated with clinical cases more often than other STs [irulence ,40. Viruironment . The virce genes . In our  to cell . LPI-1 i strains ,41. Thisdherence . How thether STs ,41.L. monocytogenes strains of animal and food origin. Resistance, e.g., to penicillin, ampicillin, gentamycin, streptomycin, tetracycline, and trimethoprim-sulfamethoxazole has been reported [fosX and lin genes were detected. These two genes were present in all 55 L. monocytogenes isolates. Earlier studies have shown fosX and lin to be present in nearly all L. monocytogenes isolates [L. monocytogenes strains [L. monocytogenes of game and game meat origin found in Finland are so far sensitive to antimicrobials. One explanation may be that hunted game in Finland have no access to feed contaminated with resistant L. monocytogenes strains.AMR is a serious public health issue due to increasing resistance. There is also a trend of increasing AMR among reported ,43. Howeisolates . This ca strains ,43. Our L. monocytogenes isolates of game origin using tools available on open-source platforms and Ridom software. Our study demonstrates that game meat is contaminated with various STs associated with human listeriosis. All L. monocytogenes isolates were potentially pathogenic, carrying most important virulence genes. No acquired AMR genes were found, indicating that all isolates were sensitive to most of the important antimicrobials used to treat listeriosis. Some of the isolates from mallard and deer meat belonged to CLs that were formed by very closely related isolates, indicating common contamination sources. Contaminated game meat may pose a public health problem, and game meat should therefore be handled and stored correctly.In this study, we analysed the sequence data of


Micrococcus yunnanensis (M. yunnanensis) is an endophytic actinomycete that was originally isolated from the roots of Polyspora axillaris in 2009, and no human infections caused by this organism have yet been reported. We report the first case of community-acquired pneumonia caused by M. yunnanensis and propose that M. yunnanensis should be considered as an emerging pathogen in medical practice. A 30-year-old woman was admitted to our hospital with fever, paroxysmal dry cough with sputum, and pharyngalgia. Laboratory tests revealed an increase in several inflammatory indicators, and a computerized tomography scan of the chest showed scattered infection foci in both lungs. Bronchoalveolar lavage fluid was collected via bronchoscopy for microbial culture and pathological examination.M. yunnanensis by 16S rRNA gene sequencing. The patient was diagnosed with community-acquired pneumonia based on the diagnostic criteria.The isolate from bronchoalveolar lavage fluid was identified as The patient was treated with intravenous amoxicillin/clavulanate potassium, levofloxacin hydrochloride tablets, and compound methoxyphenamine capsules on the day after admission. After 3 days of treatment, the patient’s physiological conditions and inflammatory indicators normalized, and 6-month follow-up showed no abnormalities.M. yunnanensis is unclear, the present case indicates an emerging pathogen in medical practice. MALDI-TOF MS has a limited ability to identify novel or rare pathogenic species, and 16S rRNA gene sequencing is of great value in some circumstance.Although the pathogenicity of  Subsequent studies have reported that M. yunnanensis was also isolated from Catharanthus roseus and pharmaceutical sewage.,3 Recently, Lee et al reported the complete genome sequence of M. yunnanensis TT9 obtained from the skin of the forehead of a healthy volunteer.M. yunnanensis has been characterized as a nonpathogenic organism that shows no external signs of infection in its host. Most studies have focused on the role of M. yunnanensis in industrial wastewater treatment and plant growth promoting activities,–7 little is known about its role in the medical field. Here, we describe a case of human infection caused by M. yunnanensis isolated from bronchoalveolar lavage fluid (BALF) of a patient diagnosed with community-acquired pneumonia.Micrococcus 9/L (83.9% neutrophils), platelet count of 207 × 109/L , an erythrocyte sedimentation rate of 31 mm/h , hypersensitive C-reactive protein (whole blood) of 2.69 mg/dL , total protein of 63.5 g/L , and albumin of 38.1 g/L . Tests for SARS-CoV-2, Mycoplasma pneumoniae, and Chlamydia pneumoniae were negative. A computerized tomography scan of the chest showed scattered infection foci in both lungs  and did not quit smoking. Physical examination revealed a sick female, with a blood pressure of 104/68 mm Hg, temperature of 38.5°C, pulse of 91 beats/min, and respiratory rate of 20 breaths/min. Laboratory tests revealed the following: white blood cell count of 14.1 × 102. After incubation for 18-24h, isolated strains with yellow, smooth, and circular colonies on Columbia blood agar plates were obtained and named BL3003. However, the strain BL3003 could not be classified into species using matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) . Subsequently, 16S rRNA gene sequencing was conducted to classify this strain using universal 16S rRNA primers . The 16S rRNA sequence of the strain BL3003 was analyzed using the Basic Local Alignment Search Tool in the GenBank database (https://blast.ncbi.nlm.nih.gov). The strain BL3003 exhibited highest (99.72%) 16S rRNA gene sequence similarity to the type strain of M. yunnanensis YIM 65004T (GenBank accession no. FJ214355). Multiple alignments with sequences of the most closely related Micrococcus species and the levels of sequence similarity were calculated using CLUSTALW. A phylogenetic tree was constructed using the neighbor-joining method by using MEGA software version 11. The topology of the phylogenetic tree was evaluated using the bootstrap resampling method with 1000 replicates. The phylogenetic tree .Histochemical analysis of BALF by staining with Papanicolaou stain and Gram’s method showed a large number of neutrophils and macrophages Fig. C and gra The AST results showed that strain BL3003 was resistant to erythrocin, azithromycin, and clindamycin, and sensitive to the other different kinds of antibiotics tested in this study. The patient was diagnosed with community-acquired pneumonia and treated with intravenous (i.v.) amoxicillin/clavulanate potassium, levofloxacin hydrochloride tablets, and compound methoxyphenamine capsules on the day after admission, her body temperature returned to normal few hours later. After 3 days of treatment, the patient’s physiological conditions and inflammatory indicators were normalized. Consequently, the patient was discharged with medicines after 1 week of hospitalization, and 6-month follow-up showed no abnormalities.Antimicrobial susceptibility testing (AST) was performed using the minimum inhibitory concentration method with an XK-type automatic bacteria identification/drug sensitivity analyzer , and 18 antibiotics were tested  and M. yunnanensis should be reclassified as later heterotypic synonyms of Micrococcus luteus (M. luteus). Among these species, M. luteus is the most studied and is relevant to human health. Several cases have reported that M. luteus is the causative agent of infective endocarditis, bacteremia, and brain abscess in rare circumstance. Only one case has reported peritoneal dialysis-related peritonitis caused by M. aloeverae. We reported for the first time that M. yunnanensis was isolated from BALF of a patient with community-acquired pneumonia.The genus P. axillaris, showing no external signs of infection or negative effects on the host. Since it has never been detected in a human specimen, there have been no studies on its pathogenicity. Immunodeficiency is an important condition for opportunistic pathogens that cause infection; however, the patient in this case had no history of immunocompromised diseases or drug abuse. It is worth noting that the patient has a 7-year history of smoking that is proposed as significant risk factor for community-acquired pneumonia. According to the drug sensitivity test, it seems M. yunnanensis appears to be sensitive to most antibiotics, including amoxicillin/clavulanate and levofloxacin, which are used to treat the patient. The bacteria were efficiently eliminated upon usage of antibiotics, and the patient’s condition improved simultaneously.M. yunnanensis is an endophytic bacterium that colonizes the roots of M. aloeverae, M. yunnanensis, and M. luteus proposed by Huang et al, these 3 species belong to the M. luteus group, and M. aloeverae and M. yunnanensis were reclassified as later heterotypic synonyms of M. luteus.M. luteus and M. aloeverae have been proven to be related to human diseases; however, M. yunnanensis has not been reported. In this case, M. yunnanensis was isolated from a patient diagnosed with community-acquired pneumonia and we propose that M. yunnanensis is potentially pathogenic to human healthcare. The identification of pathogenic organisms is of great importance in the diagnosis and treatment of infectious diseases. The MALDI-TOF MS is a valuable technique for pathogen identification developed in recent years, with reduced cost and speed of execution. However, this method alone is inadequate for bacterial classification, especially for emerging pathogens such as M. yunnanensis. Multidimensional analysis, including 16S rRNA sequencing, will be valuable for accurate classification of large numbers of species within some genera, thus distinguishing strains isolated from infectious diseases and the epidemiology of each species.Based on the reclassification of M. yunnanensis in a woman without obvious immunodeficiency. The clinical isolate was identified by means of 16S rRNA gene sequencing. The present case indicates an emerging pathogen in medical practice. More cases of infections by this organism should be collected and integrated to explore the potential pathogenicity and epidemiology of M. yunnanensis.In conclusion, we reported the first case of community-acquired pneumonia caused by Conceptualization: Yingmiao Zhang.Formal analysis: Yan Jiang.Resources: Yingmiao Zhang; Yu Zhan.Supervision: Tian Qin; Zhongxin Lu.Writing—original draft: Yingmiao Zhang; Hui Wang.Writing—review and editing: Yingmiao Zhang; Zhongxin Lu.