Bacterial Production Research Articles

From pathogenesis to treatment: the impact of bacteria on cancer.

The intricate relationship between cancer and bacteria has garnered increasing attention in recent years. While traditional cancer research has primarily focused on tumor cells and genetic mutations, emerging evidence highlights the significant role of microbial communities within the tumor microenvironment in cancer development and progression. This review aims to provide a comprehensive overview of the current understanding of the complex interplay between cancer and bacteria. We explore the diverse ways in which bacteria influence tumorigenesis and tumor behavior, discussing direct interactions between bacteria and tumor cells, their impact on tumor immunity, and the potential modulation of the tumor microenvironment. Additionally, we delve into the mechanisms through which bacterial metabolites and extracellular products May affect cancer pathways. By conducting a thorough analysis of the existing literature, we underscore the multifaceted and intricate relationship between bacteria and cancer. Understanding this complex interplay could pave the way for novel therapeutic approaches and preventive strategies in cancer treatment.

PSIV-11 The inhibitory activity of phyto-phenolic compounds on ammonia-producing ruminal bacteria

Abstract Antibiotic resistance has progressively increased over time, thus necessitating the discovery of sustainable, novel antimicrobials. Ammonia producing bacteria (APB) are predominant organisms occupying many niches in the rumens of cattle and soil. The objective of this study was to discover target phyto-phenolic compounds (PPCs) that can act as replacements for existing antimicrobials, as well as at what concentration ranges may be optimal for inhibition of APB growth. In recognition of this discovery, producers may have alternative antimicrobial compounds for use in managing APB and common silage spoilage organisms. To shed light on this matter, inhibitory concentrations (IC) and ammonia assays were implemented. ICs analyzed the inhibitory action of six structurally similar PPCs: thymol, eugenol, carvacrol, hydro-cinnamic acid (H-CA), trans-cinnamic acid (T-CA), and phloroglucinol. Five APB (SR, MD1, F, BG1, and B14) were exposed to five concentrations of phytochemicals via a 10% v/v serial dilution: 10, 1, 0.1, 0.01, 0.001 mM. Following serial dilution, the bacteria were incubated ~24h at 39°C in a water bath, and visually inspected for growth. Ammonia assays utilized the least, fully inhibitory concentration of each phytochemical identified via the inhibitory concentration experiments. Ammonia concentrations were determined using the phenolic acid/hypochlorite method. All protocols utilized anaerobic sterile technique. All experiments were repeated in triplicate and significant results (P < 0.05) were determined using a one-way ANOVA with Tukey’s post hoc test utilizing OriginPro statistical software. Inhibition results revealed that eugenol, thymol, and carvacrol were successful in inhibiting bacterial growth at a concentration range of 10 mM to 1 mM. However, phloroglucinol was not fully inhibitory at any concentration tested against any bacteria. Eugenol, thymol, carvacrol were fully inhibitory at either 10 mM or 1 mM for all organisms tested except BG1, where it was only inhibitory at 10 mM. Ammonia analyses determined that eugenol and carvacrol greatly reduced ammonia production for all species tested except for B14, a generalist, which had no measurable changes in ammonia for any of the treatments including control. Thymol reduced ammonia production for MD1, BG1, and SR. Additionally, H-CA and T-CA decreased ammonia production of SR compared with control but not as much as eugenol or carvacrol. Results suggested that the growth of APB was generally inhibited by the greatest concentrations of PPCs (10 mM to 1 mM) tested here. Additionally, phloroglucinol did not inhibit bacterial growth therefore it was not included in the ammonia analyses. Eugenol and carvacrol significantly reduced ammonia production in all organisms, therefore indicating possible antimicrobial candidates to be further investigated. In comparison with carvacrol and eugenol, T-CA and H-CA made a modest to negligible reduction in ammonia. Phyto-phenolic compounds that were successful in inhibiting bacterial growth and reducing ammonia production demonstrate several promising target antimicrobials.

Characterization of toxigenic genes of Bacillus cereus strains isolated from different spices sold in Algeria.

Many cooked foods are prepared with spices and dried herbs; these can be contaminated by several types of microorganisms, including aerobic spore-forming bacteria. The Bacillus cereus group is very widespread in nature and is known among the common food contaminants. They are involved in food poisoning, causing two types of syndromes, diarrheal and emetic. The aims of the present work were to determine the prevalence of toxigenic Bacillus cereus spores in spices and herbs marketed in the Laghouat area and to identify their toxigenic genes via PCR. Among the 191 samples, 14.13% were determined to be B. cereus, with concentrations ranging from 2.52 to 5.82 log cfu/g, where the highest level of contamination was observed in allspice and ginger. Moreover, entFM (100%), nhe (88.23%) and cytK (70.58%) were the most frequently identified toxin genes, whereas hbl (23.52%) was less common, and no emetic toxin-encoding gene (cesB) was found in any of the samples. Considering the results of the present study, the B. cereus microbial load and toxin gene profiles of spices show that spices have potential for public health in Algeria. In this context, it is crucial to guarantee the microbiological safety of spices by respecting good hygiene practices, eliminating bacterial spores and toxin production via sterilization and using appropriate packaging for these products.

Intestinal fatty acid binding protein is associated with infarct size and cardiac function in acute heart failure following myocardial infarction

BackgroundIn acute heart failure (HF), reduced cardiac output, vasoconstriction and congestion may damage the intestinal mucosa and disrupt its barrier function. This could facilitate the leakage of bacterial products into...

Identification of Kibdelomycin and Related Biosynthetic Gene Clusters and Characterization of the C‐Branching of Amycolose

AbstractMany bacterial natural products contain C‐branched sugars, including components from the outer cell wall or antibiotically active metabolites. The enzymatic C‐branching of keto sugars leading to longer side chains (≥C2) is catalyzed by thiamine diphosphate (ThDP)‐dependent enzymes. Chiral tertiary α‐hydroxy ketones are formed in this process. The ThDP‐dependent enzymes that catalyze C‐branching reactions belong to one of three enzymatic superfamilies: decarboxylases, transketolases, and α‐ketoacid dehydrogenases 2, but branching of keto sugars has only been demonstrated for decarboxylases. In this study, we showed that an α‐ketoacid dehydrogenase is responsible for C‐branching of the deoxyketo sugar amycolose in the biosynthesis of kibdelomycin in Kibdelosporangium sp. MA7385. In addition, we characterized an amino transferase in the same biosynthetic gene cluster (BGC) that accepts a sterically demanding tertiary α‐hydroxy ketone in a downstream reaction. Subsequently, we identified approximately 400 similar BGCs in silico, suggesting that there is a large diversity of possible ThDP‐dependent enzymes catalyzing the C‐branching of keto sugars and subsequent modifications.

Chronic Oral Inoculation of Porphyromonas gingivalis and Treponema denticola Induce Different Brain Pathologies in a Mouse Model of Alzheimer Disease.

Periodontitis is a chronic inflammatory disease driven by dysbiosis in subgingival microbial communities leading to increased abundance of a limited number of pathobionts, including Porphyromonas gingivalis and Treponema denticola. Oral health, particularly periodontitis, is a modifiable risk factor for Alzheimer disease (AD) pathogenesis, with components of both these bacteria identified in postmortem brains of persons with AD. Repeated oral inoculation of mice with P. gingivalis results in brain infiltration of bacterial products, increased inflammation, and induction of AD-like biomarkers. P. gingivalis displays synergistic virulence with T. denticola during periodontitis. The aim of the current study was to determine the ability of P. gingivalis and T. denticola, grown in physiologically relevant conditions, individually and in combination, to induce AD-like pathology following chronic oral inoculation of female mice over 12 weeks. P. gingivalis alone significantly increased all 7 brain pathologies examined: neuronal damage, activation of astrocytes and microglia, expression of inflammatory cytokines interleukin 1β (IL-1β) and interleukin 6 and production of amyloid-β plaques and hyperphosphorylated tau, in the hippocampus, cortex and midbrain, compared to control mice. T. denticola alone significantly increased neuronal damage, activation of astrocytes and microglia, and expression of IL-1β, in the hippocampus, cortex and midbrain, compared to control mice. Coinoculation of P. gingivalis with T. denticola significantly increased activation of astrocytes and microglia in the hippocampus, cortex and midbrain, and increased production of hyperphosphorylated tau and IL-1β in the hippocampus only. The host brain response elicited by oral coinoculation was less than that elicited by each bacterium, suggesting coinoculation was less pathogenic.

Optimization of Bacterial Cellulose Production from Waste Figs by Komagataeibacter xylinus

This study aimed to use waste figs as an alternative substrate for bacterial cellulose (BC) production by Komagataeibacter xylinus and optimize the identified process parameters to maximize the concentration of BC. Among the nutrients screened by Plackett–Burman (PB) design, yeast extract was found to be significant in BC production. Response surface methodology was used to investigate the effect of fermentation parameters on BC production. A maximum BC concentration of (8.45 g/L), which is among the highest BC concentrations reported so far, was achieved at the optimum levels of fermentation variables (initial pH 6.05, initial sugar concentration 62.75 g/L, temperature 30 °C). The utilization of response surface methodology (RSM) proved valuable in both optimizing and finding the interactions between process variables during BC production. Scanning electron microscope (SEM) analysis showed a dense structure of BC, characterized by ribbon-like nanofibrils with diameters ranging from 23 to 90 nm while the attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectrum of BC confirmed that the material obtained was cellulose. The X-ray diffraction (XRD) analysis showed that the crystallinity of the BC samples was 70% for BC produced on waste fig medium and 61% for BC produced on Hestrin–Schramm (HS) medium. This is the first detailed study on the production of BC from waste figs, and the findings of this study demonstrated that waste figs can be used as an effective substrate for the production of BC.

High-throughput screening identification of apigenin that reverses the colistin resistance of mcr-1-positive pathogens.

The plasmid-mediated gene mcr-1 that makes bacteria resistant to the antibiotic colistin is spreading quickly, which means that colistin is no longer working well to treat Gram-negative bacterial infections. Herein, we utilized a computer-aided high-throughput screening drugs method to identify the natural product apigenin, a potential mcr-protein inhibitor, which effectively enhanced the antimicrobial activity of colistin. Several assays, including a checkerboard minimum inhibitory concentration assay, a time-kill assay, the combined disk test, molecular simulation dynamics, and animal infection models assay, were conducted to verify whether apigenin enhanced the ability of colistin to fight Gram-negative bacterial infections. The results showed that apigenin improved the antimicrobial activity of colistin against multidrug-resistant Enterobacteriaceae infection. Moreover, apigenin not only did not increase the toxic effect of colistin but also had the ability to effectively inhibit the frequency of bacterial resistance mutations to colistin. Studies clearly elucidated that apigenin could interfere with the thermal stability of the protein by binding to the mcr-1 protein. Additionally, the combination of apigenin and colistin could exert multiple effects, including disrupting bacterial membranes, the generation of bacterial nitric oxide and reactive oxygen species, as well as inhibiting bacterial adenosine triphosphate production. Furthermore, the addition of apigenin was able to significantly inhibit colistin-stimulated high expression levels of the bacterial mcr-1 gene. Finally, apigenin exhibited a characteristic anti-inflammatory effect while enhancing the antimicrobial activity of colistin against mcr-1-positive Escherichia coli (E. coli) infected animals. In conclusion, as a potential lead compound, apigenin is promising in combination with colistin in the future treatment of mcr-1-positive E. coli infections.IMPORTANCEThis study found that apigenin was able to inhibit the activity of the mcr-1 protein using a high-throughput virtual screening method. Apigenin effectively enhanced the antimicrobial activity of colistin against multidrug-resistant Enterobacteriaceae, including mcr-1-positive strains, in vitro and in vivo. This study will provide new options and strategies for the future treatment of multidrug-resistant pathogen infections.

Ensuring Tree Protection, Growth and Sustainability by Microbial Isolates

Antimicrobial properties of the new strains of micro-organisms isolated from natural sources of various ecological niches in the Moscow region and the Republic of Tatarstan were studied. Antifungal activity of isolates was detected in a test culture of toxin-producing microscopic fungi that can cause animal and plant diseases: Aspergillus flavus, Candida albicans, Fusarium oxysporum and Penicillium spp. Of the 46 studied micro-organisms of genera Bacillus, Lactobacillus, Lactococcus and Streptomyces isolates, there are four strains (Bacillus subtilis, Lactobacillus plantarum, Propionibacterium freudenreichii and Streptomyces spp.) that showed an ability to produce biologically active metabolites with a pronounced antimicrobial potential against phytopathogenic fungi metabolites. Based on the selected four strains, a Bacterial product LRV composition has been created. Scots pine, pedunculate oak and small-leaved linden seedlings with single and double foliar treatment and Bacterial product LRV at a concentration of 10 mL/L led to an increase in the growth of the aboveground part by 31.8, 51.9 and 25.4%, respectively, and the underground part by 25.0, 37.2 and 25.7%, respectively, compared to the control. The weight of seedlings at the end of the study exceeded the control variant by an average of 26.0, 44.0 and 78.0%, respectively. Plant protection Bacterial product LRV use did not have a significant effect on the group of molds that caused the powdery mildew and Schütte disease damage to trees. The Biological product LRV provided plant protection from fungal diseases caused by Lophodermium pinastri Chev. and Microsphaera alphitoides.

A Brief Review on the Emerging Role of Bacterial Pigments: From Industrial Uses to Novel Therapeutic Applications

Pigments produced by microorganisms are in high demand due to their safe, non-toxic, and biodegradable characteristics in both industrial and pharmaceutical fields. Using bacteria for pigment production offers several advantages, such as a short life cycle and ease of genetic modification. Numerous studies have highlighted that soil bacteria significantly contribute to the production of coloured pigments as secondary metabolites. Bacterial pigments are well-known for their antimicrobial, antioxidant, and anticancer properties, offering new therapeutic opportunities for the development of novel drugs. However, compared to fungal pigments, most bacterial pigments are still in the research and development stage. Hence, efforts to intensify bacterial pigment production are essential to make them readily available on the market at a low cost for various applications. This review article sheds light on bacterial pigments and their various applications based on the data available in the literature.

How carbon sources drive cellulose synthesis in two Komagataeibacter xylinus strains

Bacterial cellulose synthesis from defined media and waste products has attracted increasing interest in the circular economy context for sustainable productions. In this study, a glucose dehydrogenase-deficient Δgdh K2G30 strain of Komagataeibacter xylinus was obtained from the parental wild type through homologous recombination. Both strains were grown in defined substrates and cheese whey as an agri-food waste to assess the effect of gene silencing on bacterial cellulose synthesis and carbon source metabolism. Wild type K2G30 boasted higher bacterial cellulose yields when grown in ethanol-based medium and cheese whey, although showing an overall higher d-gluconic acid synthesis. Conversely, the mutant Δgdh strain preferred d-fructose, d-mannitol, and glycerol to boost bacterial cellulose production, while displaying higher substrate consumption rates and a lower d-gluconic acid synthesis. This study provides an in-depth investigation of two K. xylinus strains, unravelling their suitability for scale-up BC production.

Calcium Rescues Streptococcus pneumoniae D39 ΔmntE Manganese-Sensitive Growth Phenotype.

Calcium (Ca2+) functions as a universal signal messenger in eukaryotes but in bacteria, the physiological roles for Ca2+ are limited. Here, we examine the role of Ca2+ in Streptococcus pneumoniae during manganese (Mn2+) intoxication. S. pneumoniae mntE mutants, lacking the Mn2+ efflux transporter, exhibit impaired growth due to accumulation of Mn2+ when exposed to elevated exogenous Mn2+. This Mn2+-sensitive growth defect is restored to wild-type growth level by exogenous Ca2+, in a Ca2+-dependent manner. Despite growth restoration of the mntE mutant to wild-type levels, cellular Mn2+ remains elevated in this strain. Bacterial capsule production is also increased for the mntE mutant, resulting in reduced adherence capacity to surfaces and poor biofilm formation, which is consistent with it experiencing Mn2+ intoxication. Ca2+ presence did not significantly impact bacterial capsule production or biofilm formation. Further analysis of the cell morphology demonstrates that Ca2+ contributes to cell division and reduces cell chain lengths. Together, these data describe the first role of Ca in S. pneumoniae that has potential implications in bacterial virulence since Ca affects cell division and likely Mn2+-associated cellular processes.

Negative feedback of cyclic di-GMP levels optimizes switching between sessile and motile lifestyles in Vibrio cholerae.

The signaling molecule cyclic di-GMP (cdG) controls the switch between bacterial motility and biofilm production, and fluctuations in cellular levels of cdG have been implicated in Vibrio cholerae pathogenesis. Intracellular concentrations of cdG are controlled by the interplay of diguanylate cyclase (DGC) enzymes, which synthesize cdG to promote biofilms, and phosphodiesterase (PDE) enzymes, which hydrolyse cdG to drive motility. To track the complete regulatory logic of how V. cholerae responds to changing cdG levels, we followed a timecourse of overexpression of either the V. harveyi diguanylate cyclase QrgB or a variant of QrgB lacking catalytic activity (QrgB*). We find that QrgB increases cdG levels relative to QrgB* for 30 minutes after overexpression, but the effect of QrgB on cdG levels plateaus at 30 minutes, indicating tight adaptive control of cdG levels. In contrast, loss of VpsR, a master regulator activating biofilm formation upon binding to cdG, leads to higher baseline levels of cdG and continuously increasing cdG through 60 minutes after QrgB induction, revealing the existence of a negative feedback loop on cdG levels operating through VpsR. Through a combination of RNA polymerase ChIP-seq, RNA-seq, and genetic approaches, we show that transcription of a gene encoding a PDE, cdgC, is activated by VpsR at high cdG concentrations, mediating this negative feedback on cdG levels. We further identify a transcript encoded within, and antisense to, the cdgC open reading frame which we name sRNA negative regulator of CdgC (SnrC). RNA polymerase ChIP-seq and RNA-seq demonstrate SnrC to be expressed specifically under conditions of high cdG in the absence of VpsR. Ectopic SnrC expression increases cdG levels in a manner dependent on CdgC, demonstrating that its effect on cdG levels is likely through interference with CdgC production. Further, although cells lacking cdgC exhibit enhanced biofilm formation, these mutants are outcompeted by wild type V. cholerae in colonization assays that reward a combination of attachment, dispersal, and motility behaviors. These results underscore the importance of negative feedback regulation of cdG to maintain appropriate homeostatic levels for efficient transitioning between biofilm formation and motility, both of which are necessary over the course of the V. cholerae infection cycle.

In Silico Screening of Sulfonamide Derivatives against Glycolytic Enzymes Reveals Mutation‐Independent Interactions of Sulfisoxazole and Sulfamethazine

AbstractGlycolysis is a highly conserved biochemical process that involves the sequential conversion of glucose to pyruvate and the generation of adenosine triphosphate (ATP), generating energy for cellular functions. This is carried out by several enzymes that play an important role in glucose metabolism. The sulfonamide group of drugs is commonly used as an antibiotic that inhibits the bacterial folic acid production pathway, although its effects on glycolytic enzymes are unclear. Since altered glycolytic enzyme expression is linked to a variety of metabolic disorders as well as cancer, the quest for new ligands against glycolytic enzymes is never over. We revealed that different sulfonamide derivatives interact with glycolytic enzymes with varying binding affinities. Among the selected sulfonamides, sulfisoxazole and sulfamethazine had greater binding scores to certain glycolytic enzymes than others. In silico point mutation analysis predicts that sulfisoxazole and sulfamethazine interact with certain glycolytic enzymes in a residue‐independent manner. This research generates a repository of the 2D interactions of sulfonamide derivatives with glycolytic enzymes, binding energies, impacts of induced point mutations on the local environment, and changes in protein structural flexibility. In the future, sulfisoxazole and sulfamethazine may be repurposed as specific glycolytic enzyme inhibitors to combat a variety of glycolysis‐related metabolic disorders.

Bacterial production of ProTx-I, a gating modifier toxin that inhibits voltage-gated sodium (NaV) and TRPA1 channels

Bacterial production of ProTx-I, a gating modifier toxin that inhibits voltage-gated sodium (NaV) and TRPA1 channels

Staphylococcus aureus lipid factors modulate melanoma cell clustering and invasion.

The microbiome can influence cancer development and progression. However, less is known about the role of the skin microbiota in melanoma. Here, we took advantage of a zebrafish melanoma model to probe the effects of Staphylococcus aureus on melanoma invasion. We found that S. aureus produces factors that enhance melanoma invasion and dissemination in zebrafish larvae. We used a published in vitro 3D cluster formation assay that correlates increased clustering with tumor invasion. S. aureus supernatant increased clustering of melanoma cells and was abrogated by a Rho-Kinase inhibitor, implicating a role for Rho-GTPases. The melanoma clustering response was specific to S. aureus but not to other staphylococcal species, including S. epidermidis. Our findings suggest that S. aureus promotes melanoma clustering and invasion via lipids generated by the lipase Sal2 (officially known as GehB). Taken together, these findings suggest that specific bacterial products mediate melanoma invasive migration in zebrafish.

Isolation and Screening of Tyrosinase Producing Bacteria from Ahmednagar Region

Isolation and Screening of Tyrosinase Producing Bacteria from Ahmednagar Region

Bioengineering Approaches and Novel Biomaterials to Enhance Sternal Wound Healing after Cardiac Surgery: A Crosstalk between Innovation and Surgical Practice.

Median sternotomy and steel wires for sternal closure are the standard approach for cardiac surgery. An incomplete repair associated with chest wall motion, especially in the presence of predisposing factors, can lead to life-threatening deep sternal wound infection, also known as mediastinitis, in 2-5% of cases. Despite current antibiotic and surgical treatments, mediastinitis is associated with a 10-40% mortality rate and a significant increase in morbidity and hospital stay. High mortality and difficult treatment appear to be due to bacterial biofilm, a self-produced extracellular polymeric product that incorporates host tissue and is responsible for the failure of immune defenses and standard antimicrobial therapies. Nanostructures are an effective strategy to enhance the healing process, as they establish a favorable environment for the neosynthesis of the extracellular matrix, supporting tissue development. Synthetic polymers have been proven to exhibit suitable biodegradable and mechanical properties, and their biofunctionalization to enhance cell attachment and interaction with the extracellular matrix is being widely investigated. The use of antibiotic treatments suspended in poly-D,L-lactide and polyethylene oxide and electrospun into nanofibers, or in sponges, has been shown to inhibit bacterial biofilm production. Additionally, growth factors can be incorporated into 3D bioresorbable scaffolds with the aim of constituting a structural and biological framework to organize and expedite the healing process. Therefore, these combined approaches may change the treatment of mediastinitis in the near future.

Infection, inflammation and hepatic encephalopathy from a clinical perspective.

Hepatic encephalopathy (HE) is a syndrome that is associated with both acute and chronic liver injury. It manifests as a wide spectrum of neuropsychological abnormalities, ranging from subtle impairments in executive higher functions observed in cirrhosis, through to coma in acute liver failure. In acute liver failure, the central role of ammonia in the development of brain oedema has remained undisputed for 130 years. It latterly became apparent that infection and inflammation were profound determinants for the development of severe hepatic encephalopathy, associated with the development of cerebral oedema and intracranial hypertension. The relationship of the development of hepatic encephalopathy with blood ammonia levels in cirrhosis is less clear cut and the synergistic interplay of inflammation and infection with ammonia has been identified as being fundamental in the development and progression of hepatic encephalopathy. A perturbed gut microbiome and the presence of an impaired gut epithelial barrier that facilitates translocation of bacteria and bacterial degradation products into the systemic circulation, inducing systemic inflammation and innate and adaptive immune dysfunction, has now become the focus of therapies that treat hepatic encephalopathy in cirrhosis, and may explain why the prebiotic lactulose and rifaximin are efficacious. This review summarises the current clinical perspective on the roles of inflammation and infection in hepatic encephalopathy and presents the evidence base for existing therapies and those in development in the setting of acute and chronic liver failure.

Metabolic-methane mitigation by combination of probiotic Escherichia coli strain Nissle 1917 and biochar in rumen fluid in vitro fermentation of dairy cow

In response to climate change, there have been various trials to reduce methane emissions from ruminant animals in the livestock industry, and one of the typical candidates is probiotics used as feed additives. We employed Escherichia coli Nissle 1917 (EcN) as a promising probiotic for the reduction of methane emissions and investigated the molecular mechanism of methane-reducing effects using CRISPR/Cas9. The anaerobic culture of EcN with biochar (BC) could significantly increase acetate and hydrogen production. Additionally, RNA-seq analysis revealed an upregulation in the expression of acetate synthesis genes, namely, pta, poxB, and ackA. Subsequently, treatment of rumen fluid with EcN significantly decreased methane and increased acetate and propionate production, as observed through the analysis of short-chain fatty acids, and these effects could accelerate with additional supplementation of BC. The observation of changes in the microbial composition of rumen fluid following treatment with EcN and BC was made through rumen metagenomic analysis and RT-qPCR. The results revealed an increase in acetogen and propionate producing bacteria abundance and a decrease in methanogen abundance. Based on these findings, the CRISPR/Cas9 system was employed to elucidate the molecular mechanism of the methane-reducing capability of EcN. Gene deletion was performed targeting the genes poxB, ackA, and pta as key factors in acetate pathway in E. coli. The knockout of poxB, ackA, and pta could lead to the elimination of the methane-reduction of EcN as well as acetate-producing abilities. Collectively, the methane reduction ability of EcN in rumen fluid is associated with its acetate production capability, and the addition of BC significantly enhances methane mitigation capability of EcN.