Cell-free Culture Supernatant Research Articles

Differential alteration in Lactiplantibacillus plantarum subsp. plantarum quorum-sensing systems and reduced Candida albicans yeast survival and virulence gene expression in dual-species interaction.

Candida albicans (C. albicans) and Lactiplantibacillus plantarum subsp. plantarum (L. plantarum) are frequently identified in various niches, but their dual-species interaction, especially with C. albicans in yeast form, remains unclear. This study aimed to investigate the dual-species interaction of L. plantarum and C. albicans, including proliferation, morphology, and transcriptomes examined by selective agar plate counting, microscopy, and polymicrobial RNA-seq, respectively. Maintaining a stable and unchanged growth rate, L. plantarum inhibited C. albicans yeast cell proliferation but not hyphal growth. Combining optical microscopy and atomic force microscopy, cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed during dual-species interaction. Reduced C. albicans yeast cell proliferation in mixed culture was partially due to L. plantarum cell-free culture supernatant but not the acidic environment. Upon polymicrobial transcriptomics analysis, interesting changes were identified in both L. plantarum and C. albicans gene expression. First, two L. plantarum quorum-sensing systems showed contrary changes, with the activation of lamBDCA and repression of luxS. Second, the upregulation of stress response-related genes and downregulation of cell cycle, cell survival, and cell integrity-related pathways were identified in C. albicans, possibly connected to the stress posed by L. plantarum and the reduced yeast cell proliferation. Third, a large scale of pathogenesis and virulence factors were downregulated in C. albicans, indicating the potential interruption of pathogenic activities by L. plantarum. Fourth, partial metabolism and transport pathways were changed in L. plantarum and C. albicans. The information in this study might aid in understanding the behavior of L. plantarum and C. albicans in dual-species interaction.IMPORTANCEThe anti-Candida albicans activity of Lactiplantibacillus plantarum has been explored in the past decades. However, the importance of C. albicans yeast form and the effect of C. albicans on L. plantarum had also been omitted. In this study, the dual-species interaction of L. plantarum and C. albicans was investigated with a focus on the transcriptomes. Cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed. Upon polymicrobial transcriptomics analysis, interesting changes were identified, including contrary changes in two L. plantarum quorum-sensing systems and reduced cell survival-related pathways and pathogenesis determinants in C. albicans.

Characterization of Antibiotic-Resistance Antarctic Pseudomonas That Produce Bacteriocin-like Compounds.

In this study, bacterial isolates C1-4-7, D2-4-6, and M1-4-11 from Antarctic soil were phenotypically and genotypically characterized, and their antibacterial spectrum and that of cell-free culture supernatant were investigated. Finally, the effect of temperature and culture medium on the production of antimicrobial compounds was investigated. The three bacteria were identified as different strains of the genus Pseudomonas. The three bacteria were multi-drug resistant to antibiotics. They exhibited different patterns of growth inhibition of pathogenic bacteria. M1-4-11 was remarkable for inhibiting the entire set of pathogenic bacteria tested. All three bacteria demonstrated optimal production of antimicrobial compounds at 15 °C and 18 °C. Among the culture media studied, Nutrient broth would be the most suitable to promote the production of antimicrobial compounds. The thermostability exhibited by the antimicrobial molecules secreted, their size of less than 10 kDa, and their protein nature would indicate that these molecules are bacteriocin-like compounds.

A host-microbial metabolite interaction gut-on-a-chip model of the adult human intestine demonstrates beneficial effects upon inulin treatment of gut microbiome

Background: The gut and its microbiome have a major impact on many aspects of health and are therefore also an attractive target for drug- or food-based therapies. Here, we report on the added value of combining a microbiome screening model, the i-screen, with fresh intestinal tissue explants in a microfluidic gut-on-a-chip model, the Intestinal Explant Barrier Chip (IEBC). Methods: Adult human gut microbiome (fecal pool of 6 healthy donors) was cultured anaerobically in the i-screen platform for 24 h, without and with exposure to 4 mg/mL inulin. The i-screen cell-free culture supernatant was subsequently applied to the luminal side of adult human colon tissue explants (n = 3 donors), fixed in the IEBC, for 24 h and effects were evaluated. Results: The supplementation of the media with inulin promoted the growth of Anaerostipes , Bifidobacterium , Blautia , and Collinsella in the in vitro i-screen, and triggered an elevated production of butyrate by the microbiota. Human colon tissue exposed to inulin-treated i-screen cell-free culture supernatant or control i-screen cell-free culture supernatant with added short-chain fatty acids (SCFAs) showed improved tissue barrier integrity measured by a 28.2%-34.2% reduction in FITC-dextran 4000 (FD4) leakage and 1.3 times lower transport of antipyrine. Furthermore, the release of pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α was reduced under these circumstances. Gene expression profiles confirmed these findings, but showed more profound effects for inulin-treated supernatant compared to SCFA-supplemented supernatant. Conclusion: The combination of i-screen and IEBC facilitates the study of complex intestinal processes such as host-microbial metabolite interaction and gut health.

Antivirulence effects of cell-free culture supernatant of endophytic bacteria against grapevine crown gall agent, Agrobacterium tumefaciens, and induction of defense responses in plantlets via intact bacterial cells

BackgroundCrown gall disease caused by Agrobacterium tumefaciens is a very destructive affliction that affects grapevines. Endophytic bacteria have been discovered to control plant diseases via the use of several mechanisms. This research examined the potential for controlling crown gall by three endophytic bacteria that were previously isolated from healthy cultivated and wild grapevines including Pseudomonas kilonensis Ba35, Pseudomonas chlororaphis Ba47, and Serratia liquefaciens Ou55.ResultAt various degrees, three endophytic bacteria suppressed the populations of A. tumefaciens Gh1 and greatly decreased the symptoms of crown gall. Furthermore, biofilm production and motility behaviors of A. tumefaciens Gh1were greatly inhibited by the Cell-free Culture Supernatant (CFCS) of endophytic bacteria. According to our findings, CFCS may reduce the adhesion of A. tumefaciens Gh1 cells to grapevine cv. Rashe root tissues as well as their chemotaxis motility toward the extract of the roots. When compared to the untreated control, statistical analysis showed that CFCS significantly reduced the swimming, twitching, and swarming motility of A. tumefaciens Gh1. The findings demonstrated that the endophytic bacteria effectively stimulated the production of plant defensive enzymes including superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), and total soluble phenols at different time intervals in grapevine inoculated with A. tumefaciens Gh1. The Ba47 strain markedly increased the expression levels of defense genes associated with plant resistance. The up-regulation of PR1, PR2, VvACO1, and GAD1 genes in grapevine leaves indicates the activation of SA and JA pathways, which play a role in enhancing resistance to pathogen invasion. The results showed that treating grapevine with Ba47 increased antioxidant defense activities and defense-related gene expression, which reduced oxidative damage caused by A. tumefaciens and decreased the incidence of crown gall disease.ConclusionThis is the first study on how A. tumefaciens, the grapevine crown gall agent, is affected by CFCS generated by endophytic bacteria in terms of growth and virulence features. To create safer plant disease management techniques, knowledge of the biocontrol processes mediated by CFCS during microbial interactions is crucial.

Postbiotic potential of Bacillus velezensis KMU01 cell-free supernatant for the alleviation of obesity in mice

Attention toward the preventive effects of postbiotics on metabolic diseases has increased because of greater stability and safety over probiotics. However, studies regarding the bioactive effects of postbiotics, especially from probiotic Bacillus strains, are relatively limited. The anti-obesity effects of the cell-free culture supernatant of Bacillus velezensis KMU01 (CFS–B.vele) were evaluated using high-fat-diet (HFD)-induced mice. HFD-induced mice (n = 8 per group) received equal volumes of (1) CFS-B.vele (114 mg/kg) in PBS, (2) Xenical in PBS, or (3) PBS alone by oral gavage daily for 13 weeks. The results demonstrated that CFS-B.vele changed the gut microbiota and showed anti-obesity effects in HFD-induced obese mice. The elevated Firmicutes/Bacteroidota ratio induced by HFD was decreased in the CFS-B.vele group compared to the other groups (p < 0.05). The CFS-B.vele intervention led to the enrichment of SCFA-producers, such as Roseburia and Eubacterium, in the cecum, suggesting their potential involvement in the amelioration of obesity. Due to these changes, the various obesity-related biomarkers (body weight, fat in tissue, white adipose tissue weight and size, serum LDL-cholesterol level, hepatic lipid accumulation, and adipogenesis/lipogenesis-related gene/protein expression) were improved. Our findings suggest that CFS-B.vele has potential as a novel anti-obesity agent through modulation of the gut microbiota.

Xylanase Production by Cellulomonas phragmiteti Using Lignocellulosic Waste Materials

Lignocellulosic biomass holds promise as a renewable feedstock for various applications, but its efficient conversion requires cost-effective degradation strategies. The main objective of this study was to investigate the effect of the growth conditions of Cellulomonas phragmiteti in the production of (hemi)cellulosic supernatants. To meet this aim, different lignocellulosic residues were used as carbon sources for growth using defined mineral or nutritive culture media. Cell-free culture supernatants with xylanolytic activity were produced in all the conditions evaluated, but the highest xylanase activity (15.3 U/mL) was achieved in Luria–Bertani (LB) medium containing 1% waste paper. Under these conditions, almost negligible β-glucosidase, cellobiohydrolase, β-xylosidase, and α-arabinofuranosidase activity was detected. The xylanolytic supernatant showed tolerance to salt and displayed maximal catalytic efficiency at pH 6 and 45 °C, along with good activity in the ranges of 45–55 °C and pH 5–8. As it showed good stability at 45 °C, the supernatant was employed for the hydrolysis of birchwood xylan (50 g/L) under optimal conditions, releasing 10.7 g/L xylose in 72 h. Thus, C. phragmiteti was found to produce a xylanolytic enzymatic supernatant efficiently by utilizing the cheap and abundant lignocellulosic residue of waste paper, and the produced supernatant has promising attributes for industrial applications.

Lactiplantibacillus plantarum postbiotic protects against Salmonella infection in broilers via modulating NLRP3 inflammasome and gut microbiota

Salmonella infection is a major concern in poultry production which poses potential risks to food safety. Our previous study confirmed that Lactiplantibacillus plantarum (LP) postbiotic exhibited a strong antibacterial capacity on Salmonella in vitro. This study aimed to investigate the beneficial effects and underlying mechanism of LP postbiotic on Salmonella-challenged broilers. A total of 240 one-day-old male yellow-feathered broilers were pretreated with 0.8% deMan Rogosa Sharpe (MRS) medium or 0.8% LP postbiotic (LP cell-free culture supernatant, LPC) in drinking water for 28 d, and then challenged with 1×109 CFU Salmonella enterica serovar Enteritidis (SE). Birds were sacrificed 3 d postinfection. Results showed that LPC maintained the growth performance by increasing body weight (BW), average daily gain (ADG), and average daily feed intake (ADFI) in broilers under SE challenge. LPC significantly attenuated SE-induced intestinal mucosal damage. Specifically, it decreased the intestinal injury score, increased villus length and villus/crypt, regulated the expression of intestinal injury-related genes (Villin, matrix metallopeptidase 3 [MMP3], intestinal fatty acid-binding protein [I-FABP]), and enhanced tight junctions (zona occludens-1 [ZO-1] and Claudin-1). SE infection caused a dramatic inflammatory response, as indicated by the up-regulated concentrations of interleukin (IL)-1β, IL-6, TNF-α, and the downregulation of IL-10, while LPC pretreatment markedly reversed this trend. We then found that LPC inhibited the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome by decreasing the gene expression of Caspase-1, IL-lβ, and IL-18. Furthermore, LPC suppressed NLRP3 inflammasome activation by inhibiting nuclear factor-kappa B (NF-κB) signaling pathway (the reduced levels of toll-like receptor 4 [TLR4], myeloid differentiation factor 88 [MyD88], and NF-κB). Finally, our results showed that LPC regulated gut microbiota by enhancing the percentage of Ligilactobacillus and decreasing Alistipes and Barnesiella. In summary, we found that LP postbiotic was effective to protect broilers against Salmonella infection, possibly through suppressing NLRP3 inflammasome and optimizing gut microbiota. Our study provides the potential of postbiotics on prevention of Salmonella infection in poultry.

Coproduction of bio-microbicide and silver nano-microbicide mediated by endospore-forming Bacillus and their synergetic control of plant disease

BackgroundOne-pot synthesis of metal nanoparticles under ambient temperature and pressure using reducing and stabilizing materials from microbes is energy-effective and ecofriendly, but upstream extraction of biological raw materials and downstream purification of nanoparticles from bioreactors are laborious and expensive. To simplify the productive process for using metal nanoparticles as microbicides to control plant pathogens, we use an endospore-forming Bacillus biocontrol agent to produce the nano-microbicide and use the bacterial raw materials as bio-microbicides together with the nano-microbicide.ResultsBacillus cells at the stationary phase form endospores and tolerate Ag+ and Ag nanoparticles (AgNPs), while the cell-free culture supernatant (CFCS) mediates the synthesis of AgNPs. AgNPs produced from the Bacillus culture and CFCS show similar physical, chemical, and electrical characteristics, and bactericidal and anti-biofilm activities. Moreover, the diluted products effectively protect the kiwifruit leaves from the infection of the pathogen Pseudomonas syringae pv. actinidiae.ConclusionsThis coproduction of bio-microbicide and nano-microbicide is a totally green one-pot synthesis process without extraction and purification and without production of waste and can be easily scaled up using the existing fermentation processing of Bacillus biocontrol agents. The application of the synergistic bio-microbicide and nano-microbicide can effectively control the bacterial canker disease of kiwifruit plants.Graphical

High levels of short-chain fatty acids secreted by Candida albicans hyphae induce neutrophil chemotaxis via free fatty acid receptor 2.

Candida albicans belongs to our commensal mucosal flora and in immune-competent individuals in the absence of epithelial damage, this fungus is well tolerated and controlled by our immune defense. However, C. albicans is an opportunistic microorganism that can cause different forms of infections, ranging from superficial to life-threatening systemic infections. C. albicans is polymorphic and switches between different phenotypes (e.g. from yeast form to hyphal form). C. albicans hyphae are invasive and can grow into tissues to eventually reach circulation. During fungal infections, neutrophils in particular play a critical role for the defense, but how neutrophils are directed toward the invasive forms of fungi is less well understood. We set out to investigate possible neutrophil chemoattractants released by C. albicans into culture supernatants. We found that cell-free culture supernatants from the hyphal form of C. albicans induced both neutrophil chemotaxis and concomitant intracellular calcium transients. Size separation and hydrophobic sorting of supernatants indicated small hydrophilic factors as responsible for the activity. Further analysis showed that the culture supernatants contained high levels of short-chain fatty acids with higher levels from hyphae as compared to yeast. Short-chain fatty acids are known neutrophil chemoattractants acting via the neutrophil free fatty acid receptor 2. In line with this, the calcium signaling in neutrophils induced by hyphae culture supernatants was blocked by a free fatty acid receptor 2 antagonist and potently increased in the presence of a positive allosteric modulator. Our data imply that short-chain fatty acids may act as a recruitment signal whereby neutrophils can detect C. albicans hyphae.

Assays for Studying Pseudomonas aeruginosa Secreted Proteases.

Proteolytic activity plays an essential role in Pseudomonas aeruginosa adaptation and survival in challenging environments, including the infection site. Here, a short review of the eight known proteases secreted by P. aeruginosa and of the methods used to detect their activity is provided. In addition, three simple and handy methods routinely used in our laboratory to detect proteases are described in detail. In particular, the skim milk plate assay and the azocasein assay are useful for the detection of whole proteases activity in colony-growing and cell-free culture supernatants, respectively. Conversely, the Elastin Congo-red assay allows detecting the activity of the LasB elastase, the major protease secreted by P. aeruginosa, in cell-free culture supernatants.

Ligilactobacillus salivarius 7247 Strain: Probiotic Properties and Anti-Salmonella Effect with Prebiotics.

The Ligilactobacillus salivarius 7247 (LS7247) strain, originally isolated from a healthy woman's intestines and reproductive system, has been studied for its probiotic potential, particularly against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) as well as its potential use in synbiotics. LS7247 showed high tolerance to gastric and intestinal stress and effectively adhered to human and animal enterocyte monolayers, essential for realizing its probiotic properties. LS7247 showed high anti-Salmonella activity. Additionally, the cell-free culture supernatant (CFS) of LS7247 exhibited anti-Salmonella activity, with a partial reduction upon neutralization with NaOH (p < 0.05), suggesting the presence of anti-Salmonella factors such as lactic acid (LA) and bacteriocins. LS7247 produced a high concentration of LA, reaching 124.0 ± 2.5 mM after 48 h of cultivation. Unique gene clusters in the genome of LS7247 contribute to the production of Enterolysin A and metalloendopeptidase. Notably, LS7247 carries a plasmid with a gene cluster identical to human intestinal strain L. salivarius UCC118, responsible for class IIb bacteriocin synthesis, and a gene cluster identical to porcine strain L. salivarius P1ACE3, responsible for nisin S synthesis. Co-cultivation of LS7247 with SE and ST pathogens reduced their viability by 1.0-1.5 log, attributed to cell wall damage and ATP leakage caused by the CFS. For the first time, the CFS of LS7247 has been shown to inhibit adhesion of SE and ST to human and animal enterocytes (p < 0.01). The combination of Actigen prebiotic and the CFS of LS7247 demonstrated a significant combined effect in inhibiting the adhesion of SE and ST to human and animal enterocytes (p < 0.001). These findings highlight the potential of using the LS7247 as a preventive strategy and employing probiotics and synbiotics to combat the prevalence of salmonellosis in animals and humans caused by multidrug resistant (MDR) strains of SE and ST pathogens.

Uranium bioprecipitation mediated by a phosphate-solubilizing Enterobacter sp. N1-10 and remediation of uranium-contaminated soil

Uranium (U) pollution in soils is prevalent worldwide and poses a significant health risk that will require remediation approaches. However, traditional U bioreduction by sulfate reducing bacteria (SRB) are sensitive to oxygen and are not suitable for treating aerobic topsoil. Bioprecipitation of U into uranyl phosphate (UP) mediated by phosphate-solubilizing microorganism (PSM) is not affected by oxygen. In this study, PSM strains were isolated and used for U-contaminated soil remediation. Microbial metabolites and the mechanism of PSM bioprecipitation were revealed. The results showed that strain Enterobacter sp. N1-10 had the highest phosphate-solubilizing capacity (dissolved P was 409.51 ± 8.48 mg/L). Uranium bioprecipitation was investigated by culturing the bacterium in the presence of 50 mg/L U and in the cell-free culture supernatant. The results showed that strain N1–10 had a high U removal rate (99.45 ± 0.43 %) after adding 50 mg/L U to the culture medium. A yellow precipitate was immediately formed when uranyl nitrate solution was added to the cell-free culture supernatant. The analysis indicated that bacterium produced lactic acid (37.58 mg/L), citric acid (4.76 mg/L), succinic acid (2.03 mg/L), and D-glucuronic acid (1.94 mg/L); the four organic acids solubilized Ca3(PO4)2 to form stable uranyl phosphate precipitate. The application of strain N1-10 and Ca3(PO4)2 significantly decreased the bioavailability of soil U (43.54 ± 0.52 %). In addition, pot experiments showed that PSM N1-10 and Ca3(PO4)2 promoted plant growth and markedly reduced U accumulation by pakchoi. These results demonstrate that PSM N1-10 and Ca3(PO4)2 exhibit a great potential for U bioremediation.

Staphylococcus aureus delta toxin modulates both extracellular membrane vesicle biogenesis and amyloid formation.

Extracellular membrane vesicles (MVs) produced by Staphylococcus aureus in planktonic cultures encapsulate a diverse cargo of bacterial proteins, nucleic acids, and glycopolymers that are protected from destruction by external factors. δ-toxin, a member of the phenol soluble modulin family, was shown to be critical for MV biogenesis. Amyloid fibrils co-purified with MVs generated by virulent, community-acquired S. aureus strains, and fibril formation was dependent on expression of the S. aureus δ-toxin gene (hld). Mass spectrometry data confirmed that the amyloid fibrils were comprised of δ-toxin. Although S. aureus MVs were produced in vivo in a localized murine infection model, amyloid fibrils were not observed in the in vivo setting. Our findings provide critical insights into staphylococcal factors involved in MV biogenesis and amyloid formation.

Anticancer effects of cell-free culture supernatant of Escherichia coli in bladder cancer cell line: New insight into the regulation of inflammation

Bladder cancer (BC) is the 10th most common malignancy in worldwide, with substantial mortality and morbidity if not treated effectively. According to various research, inflammatory circumstances majorly impact the microenvironment of bladder cancer, and the chronic presence of cytokines and chemokines promotes tumor progression. In this investigation, we explored the impact of cell-free culture supernatant ofEscherichia colistrain 536 on inflammatory cytokines and chemokines in bladder cancer model microarray data (GSE162251). Then we examined in silico outcomes on human bladder cancer cell line 5637 to verify and extrapolate findings. This investigation revealed for the first time that this compound has potent suppressor effects on interleukin 1 beta (IL-1β), C–C motif chemokine ligand 2 (CCL2), and C-X3-C motif chemokine ligand 1 (CX3CL1) gene expression as well as increased NAD(P)H quinone dehydrogenase 1 (NQO1), as an anti-oxidant agent, gene expression in 4, 8, and 24 h. Moreover, we confirmed that c-MYC, a member of the MYC proto-oncogene family, gene expression reduced in 5637 cells in 4 h and then followed up its expression in 8 and 24 h. In addition, our investigation demonstrated that the supernatant raised the BCL2-Associated X Protein/B-cell lymphoma 2 (BAX/BCL2) ratio, and subsequent flow cytometry analysis demonstrated that the supernatant induction apoptosis and necrosis. In conclusion, our findings demonstrate that this compound is a potential candidate for the suppression of bladder cancer progression.

Pseudomonas PA14H7: Identification and Quantification of the 7-Hydroxytropolone Iron Complex as an Active Metabolite against Dickeya, the Causal Agent of Blackleg on the Potato Plant.

Soft rot Pectobacteriaceae (SRP), such as Pectobacterium and Dickeya, are phytopathogenic agents responsible for blackleg disease on several crops, such as potatoes, affecting the yield and depressing the seed production quality. However, neither conventional nor biocontrol products are available on the market to control this disease. In this study Pseudomonas PA14H7, a bacteria isolated from potato rhizosphere, was selected as a potential antagonist agent against Dickeya solani. In order to understand the mechanism involved in this antagonism, we managed to identify the main active molecule(s) produced by PA14H7. Cell-free supernatant (CFS) of PA14H7 cultures were extracted and analyzed using LC-MS, GC-MS, and NMR. We further correlated the biological activity against Dickeya solani of extracted CFS-PA14H7 to the presence of 7-hydroxytropolone (7-HT) complexed with iron. In a second time, we have synthesized this molecule and determined accurately using LC-UV, LC-MS, and GC-MS that, after 48 h incubation, PA14H7 released, in its CFS, around 9 mg/L of 7-HT. The biological activities of CFS-PA14H7 vs. synthetic 7-HT, at this concentration, were evaluated to have a similar bacteriostatic effect on the growth of Dickeya solani. Even if 7-HT is produced by other Pseudomonas species and is mostly known for its antibacterial and antifungal activities, this is the first description of its involvement as an effective molecule against pectinolytic bacteria. Our work opens the way for the comprehension of the mode of action of PA14H7 as a biocontrol agent against potato blackleg.

Modulation of ethylene and ROS-scavenging enzymes by multifarious plant growth-promoting endophytes in tomato (Solanum lycopersicum) plants to combat Xanthomonas -induced stress

The purpose of the current study was to explore root endophytes- Priestia megaterium T3 and Bacillus cereus T4 from Moringa olefiera for the suppression of leaf spot disease in tomato plants challenged with Xanthomonas vesicatoria. Both strains had plant growth-stimulating characteristics including auxin production, solubilization of inorganic phosphate and zinc complexes, and production of ammonia, siderophore, as well as hydrolytic enzymes. An agar well diffusion and fluorescence viability assay have validated the antibacterial effect of the cell-free culture supernatant of strains T3 and T4. Liquid chromatography-mass spectrometry (LC-MS) profiling has identified the secondary metabolites in the cell-free supernatant of strains T3 and T4. The bio-priming of tomato seeds with a consortium of T3 and T4 strains has significantly declined ethylene (by 0.61-fold) and hydrogen peroxide (H2O2, 0.64-fold) concentration thus, maintaining a lower content of ROS-induced malondialdehyde (MDA, 0.91-fold) as compared to control counterparts. Consequently, the leaf spot disease severity was reduced by ∼70% in consortium-treated tomato plants in contrast to their pathogen-challenged control. The consortia (T3+T4) treatment has facilitated induced systemic resistance by enhancing enzymatic activities of phenylalanine ammonia-lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), catalase (CAT), and ascorbate oxidase (AO) to detoxify the excessive Xanthomonas-induced ROS accumulation in tomato plants. Conclusively, bacterial endophytes modulate X. vesicatoria-induced ROS response and ethylene levels in tomato plants. The current findings indicate that plant growth-promoting endophytic bacterial strains hold the potential to sustainably enhance plant growth and suppress bacterial leaf spot disease in tomato plants.

Genome and Transcriptome Analysis to Elucidate the Biocontrol Mechanism of Bacillus amyloliquefaciens XJ5 against Alternaria solani.

Early blight, caused by Alternaria solani, is an important disease affecting tomatoes. Biological control offers an environmentally friendly approach to controlling pathogens. Herein, we identified a B. amyloliquefaciens strain XJ5 and investigated its biocontrol mechanism against A. solani. A. solani growth was significantly inhibited by XJ5, with the inhibition rate of cell-free culture supernatants reaching 82.3%. Furthermore, XJ5 crude protein extracts inhibited conidia germination and altered the mycelial morphology of A. solani. To uncover the potential biocontrol mechanism of XJ5, we analyzed its genome sequence and transcriptome. The genome of XJ5 comprised a 4.16 Mb circular chromosome and two circular plasmids. A total of 13 biosynthetic gene clusters and 127 genes encoding hydrolases were identified, suggestive of the ability of XJ5 to secrete antagonistic secondary metabolites and hydrolases. Transcript analysis revealed 174 differentially expressed genes on exposing A. solani to XJ5 crude protein extracts. The expression of genes related to chitin and mannose synthesis was downregulated, indicating that XJ5 metabolites may impact chitin and mannose synthesis in A. solani. Overall, these findings enhance our understanding of the interactions between B. amyloliquefaciens and phytopathogens and pave the way for the agricultural application of this promising biocontrol agent.

3-Hydroxy coumarin demonstrates anti-biofilm and anti-hyphal efficacy against Candida albicans via inhibition of cell-adhesion, morphogenesis, and virulent genes regulation

Candida albicans, a common fungus of human flora, can become an opportunistic pathogen and causes invasive candidiasis in immunocompromised individuals. Biofilm formation is the prime cause of antibiotic resistance during C. albicans infections and treating biofilm-forming cells is challenging due to their intractable and persistent nature. The study intends to explore the therapeutic potential of naturally produced compounds by competitive marine bacteria residing in marine biofilms against C. albicans biofilm. To this end, 3-hydroxy coumarin (3HC), a compound identified from the cell-free culture supernatant of the marine bacterium Brevundimonas abyssalis, was found to exhibit anti-biofilm and anti-hyphal activity against both reference and clinical isolates of C. albicans. The compound demonstrated significant inhibitory effects on biofilms and impaired the yeast-to-hyphal transition, wrinkle, and filament morphology at the minimal biofilm inhibitory concentration (MBIC) of 250 µg mL−1. Intriguingly, quantitative PCR analysis of 3HC-treated C. albicans biofilm revealed significant downregulation of virulence genes (hst7, ume6, efg1, cph1, ras1, als1) associated with adhesion and morphogenesis. Moreover, 3HC displayed non-fungicidal and non-toxic characteristics against human erythrocytes and buccal cells. In conclusion, this study showed that marine biofilms are a hidden source of diverse therapeutic drugs, and 3HC could be a potent drug to treat C. albicans infections.

Antimicrobial activity of probiotic Streptococcus salivarius LAB813 on in vitro cariogenic biofilms

ObjectiveTo investigate the antimicrobial activity of a novel commensal strain of Streptococcus salivarius, LAB813, against Streptococcus mutans biofilms. MethodsThe inhibitory activity of LAB813 towards S. mutans was tested using mono-, dual-, and multi-species cariogenic biofilms formed on three types of orthodontic appliances (metal, ceramic, aligner). The activity of the commercially available probiotic, BLIS M18™ was used as control. ResultsLAB813 significantly inhibited S. mutans biofilms with cell killing approximating 99% for all materials. LAB813 showed effectiveness at inhibiting S. mutans in more complex multi-species biofilms with cell killing approximating 90% for all three materials. When comparing the killing kinetics of the probiotics, LAB813 had a faster rate of killing biofilms than M18. Experiments conducted with cell-free culture supernatant confirmed the presence of an inhibitory substance of proteinaceous nature. The addition of xylitol, a common sugar substitute used for human consumption, potentiated the inhibitory effects of LAB813 against S. mutans embedded in a more complex fungal-bacterial biofilm. ConclusionsLAB813 possesses strong antimicrobial activity, potent anti-biofilm properties, and enhanced antimicrobial activity in the presence of xylitol. The identification and characterization of strain LAB813 exhibiting antimicrobial activity towards S. mutans hold exciting promise for this novel strain to be developed as an oral probiotic for use in the prevention of dental caries.

Exploration of the protein-dependent mechanism of Lactobacillus crispatus GAI98322 to prevent recurrent cystitis

ObjectivesTo elucidate the mechanism of Lactobacillus crispatus (L. crispatus) suppositories to prevent patients from recurrent cystitis (RC), independent from viable-Lactobacilli-bacteria- and acid-dependent ones such as hydrogen peroxide and lactate. MethodsWe used the GAI98322 strain of L. crispatus in all experiments and pH-matched.cell-free culture supernatant of L. crispatus (CFCS) was collected. The growth inhibitory activity and the biofilm formation inhibitory activity of the CFCS against uropathogenic Escherichia coli (UPEC), Extended Spectrum beta (β) Lactamase producing (ESBL+) UPEC, and Pseudomonas aeruginosa (P. aeruginosa) was assessed by agar-disk diffusion tests and crystal violet assay. Also, CFCS was subjected to mass spectrometry to specify ingredients. ResultsThe CFCS suppressed the proliferation of E. coli, ESBL + E. coli, and P. aeruginosa. Also, the CFCS at a concentration of 40% significantly impeded the biofilm formation of these three bacteria. The aggregation-promoting factor and Lysin was detected from CFCS. ConclusionsThe cell-free supernatant from the GAI98322 strain of L. crispatus inhibits the growth/biofilm formation of broad pathogens by aggregation promoting factor and lysin, which may prevent hosts from RC regardless of the antimicrobial resistance of the pathogens and even under pH modulation.