Sterile Supernatant Research Articles

Klebsiella pneumoniae Isolated from Critically Ill Patient with Nosocomial Pneumonia Elicits a Proteinopathy

Abstract Introduction/Objective Gram-negative opportunist Klebsiella pneumoniae has been identified by the World Health Organization as a critical threat partially due to its prevalence in nosocomial pneumonia. Survivors often suffer increased long-term morbidity and mortality secondary to end-organ damage, including impaired cognition. Pseudomonas aeruginosa lung infection generates an endothelial-derived tauopathy, yet whether K. pneumoniae infection elicits this phenomenon is unknown. We hypothesize K. pneumoniae infection triggers pathogenic tau release from endothelial cells, contributing to an infection-induced proteinopathy. Methods/Case Report We utilized wildtype, control, and tau-/- pulmonary microvascular endothelial cells (PMVECs). Tau was monitored via immunoblotting. K. pneumoniae (Kp 1-008) was isolated from the bronchoalveolar lavage of a critically ill patient diagnosed with a monomicrobial nosocomial pneumonia. Supernatants were filter-sterilized and boiled prior to transfer to naïve cells to determine transmissibility. Barrier permeability was assessed via transwell assays, cell viability through resazurin, and amyloid burden was monitored with Congo Red and Thioflavin T. Results (if a Case Study enter NA) Kp 1-008 infection induced permeability and tau release from control cells in a dose-dependent manner. Tau and amyloid burden increased over time in supernatants from control PMVECs. Sterile infection supernatants from control cells increased permeability and impaired oxidative metabolism in naïve cells whereas supernatants from tau-/- cells were markedly less pathogenic. Kp 1-008 induced proteinopathic cytotoxicity persisted strongly through two passages when derived from control cells but one passage when produced from tau-/- PMVECs. Conclusion Our results suggest that 1) K. pneumoniae infection of PMVECs generates a lung endothelial-derived proteinopathy and 2) endothelial tau is a significant contributor to its virulence.

Primary Infection and Supernatants Generated by Pseudomonas aeruginosa ExoY Have Lasting Effects on Cyclic Nucleotide Signaling in Pulmonary Endothelial Cells

Rationale: Pseudomonas aeruginosa is an opportunistic pathogen and a common cause of hospital-acquire pneumonia. This gram-negative bacterium has an arsenal of virulence factors including the type three secretion system (TTSS), which encodes for an injection type needle and exotoxins. One of the four exotoxins, ExoY, is a promiscuous nucleotidylyl cyclase that utilizes F-actin as a cofactor to generate both canonical and non-canonical cyclic nucleotides (cAMP, cGMP and cCMP and cUMP respectively). While it is well known that activation of endogenous adenylyl and guanylyl cyclase leads to intracellular increases in cAMP and cGMP and release into the extracellular space, whether ExoY generated cyclic nucleotides are also effluxed out of the cell into the extracellular space is not known. In addition, it is unclear whether presence of the bacteria themselves affects endogenous cAMP signaling. Further, infection with P. aeruginosa expressing ExoY generates virulent cytotoxic amyloids from lung endothelium; however, whether these cytotoxic amyloids disrupt agonist-induced endogenous cAMP generation is unknown. Thus, we tested the hypothesis that during primary infection, cAMP and cGMP are effluxed into the extracellular space, while the primary infection itself and sterile supernatants from the primary infection attenuate isoproterenol induce increases in cAMP in pulmonary microvascular endothelial cells (PMVECs). Methods: During primary infection, PMVECs were inoculated with PBS (vehicle control) or 3 different strains of P. aeruginosa at an moi 20:1. Strains ExoY+ and ExoYK81M have a fully functional TTSS and express either catalytically active or inactive ExoY, respectively, while a third strain, ExoYTM, is unable to translocate ExoY into eukaryotic cells. During the last 15 minutes of the 4-hour infection, cells were stimulated with vehicle or isoproterenol (1 μM) and rolipram (10 μM) and cAMP and cGMP measured in the lysate and media. In separate experiments, supernatants from a primary infection were generated using the same three strains of bacteria at moi 20:1 and 4 hours after inoculation, the supernatants collected and sterilized using a 0.2 μm filter. Sterilized supernatants were added to naïve PMVECs and during the last 15 minutes of the 4-hour inoculation, cells stimulated with vehicle or isoproterenol (1 μM) and rolipram (10 μM) and cAMP and cGMP measured in the lysate and media. Results: Cyclic AMP and cGMP were elevated in both the lysate and media of PMVECs following 4-hour infection with ExoY+ and the total isoproterenol and rolipram-stimulated cAMP response was attenuated by the presence of bacteria, ExoYK81M and ExoYTM, and further blunted with ExoY+ infection. Further, inoculation with ExoYK81M and ExoYTM supernatants attenuated the total isoproterenol and rolipram-stimulated cAMP response, which was further blunted by ExoY+ supernatants. Conclusions: Overall, we demonstrate PMVECs release a large cAMP and cGMP pool into the extracellular space and both primary infection and supernatants derived from the primary infection attenuate agonist induced elevations in cAMP. HL121513, HL66299. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Biocontrol effects of three antagonistic bacteria strains against Codonopsis pilosula wilt disease caused by Fusarium oxysporum

Codonopsis pilosula wilt disease caused by Fusarium oxysporum poses a serious threat to the commercial quality and yield of C. pilosilua crops in China. In this study, three selected isolates of antagonistic bacteria, Paenibacillus polymyxa YF, Bacillus amyliquefaciens HT, and Bacillus velezensis BQ, were evaluated for their potential ability to control C. pilosula wilt disease. All three strains could colonize the roots of C. pilosula. However, strain BQ had the strongest colonization ability, in that its colonization abundance was stable at 5.2 × 103 CFU/g. Pot experiments showed that each of these three strains may contribute to enhancing C. pilosula’s growth in terms of its plant height, root length, root fresh weight, and root dry weight. All three antagonistic strains were able to effectively suppress the mycelial growth of F. oxysporum by 45 % to 65 %, with strain YF outperforming the other two strains. Similarly, 1 % sterile supernatant filtrate of each strain was capable of reducing by 40 %∼50 % the germination of F. oxysporum conidia. Pot experiments also demonstrated that strain YF was best able to ameliorate symptoms of C. pilosula wilt disease, whereas strain BQ was best able to prevent them. Similar results were obtained from ex vivo experiments, in that the strain YF was superior to the other two strains to ameliorate symptoms, while strain BQ was superior to the other two strains to prevent them. Analysis of resistance enzyme activity revealed that both peroxidase (POD) and phenylalanine ammonia-lyase (PAL) in C. pilosula roots increased significantly, by 4.5–4.7 and 1.7–1.9 times, in response to the three bacterial strain treatments. And resistance enzyme activity of all three bacterial strain treatments were significantly higher than F. oxysporum treatment and the co-treatment group (F. oxysporum + Antagonistic bacteria treatment groups).

VARIOUS ADHERENT-INVASIVE E. COLI (AIEC) AND NON-AIEC STRAINS ISOLATED FROM CROHN’S DISEASE PATIENTS GROW DIFFERENTLY WITH RUMINOCOCCUS GNAVUS PRE-DIGESTED MUCUS AND R. GNAVUS-RELEASED PRE-DIGESTED ILEAL AND COLONIC MUCUS MONOSACCHARIDES

Abstract BACKGROUND Mucus-digesting Ruminococcus gnavus (R. gnavus), adherent-invasive E. coli (AIEC), and hydrogen sulfide (H2S) are enriched in active Crohn’s disease (CD) patients compared to healthy subjects. Dual colonization of R. gnavus and E. coli LF82 in ex-germ-free mice enhanced distal colitis and H2S production in cecal contents. R. gnavus pre-digestion of mucus enhanced the ileal CD isolate AIEC LF82 growth and H2S production in vitro. H2S enhanced LF82 adhesion and invasion both in vitro and in vivo along with related LF82 adhesion gene expression and Caco-2 cell expression of CEACAM6, a receptor for AIEC ligand FimH. Interactions between other CD-derived AIEC/non-AIEC and R. gnavus are unknown. HYPOTHESIS Different AIEC and non-AIEC may grow and produce H2S differently using mucus digested by R. gnavus, which releases mucus-derived monosaccharides and sulfur substrates. METHODS We studied CD-derived clinical isolates of AIEC 541-15 (phylotype A), 541-1 (B1), LF82 and NRG857c (B2) and non-AIEC T75 (A) and lab strain K12 (A). M9 minimal media was supplemented with porcine gastric mucin (PGM), ileal (MIM) and colonic (MCoM) mucus isolated from germ-free mice. We measured growth kinetics and H2S production by these AIEC and non-AIEC following culture with sterile supernatants of each mucin/mucus media precultured with or without R. gnavus, monosaccharides obtained by R. gnavus digestion of mucus, and several sulfur substrates in the gut added to M9 minimal media. RESULTS AIEC, especially LF82 (B2), grew better with ileal mucus aerobically and, to a lesser extent, anaerobically than non-AIEC strains, with even greater AIEC growth with R. gnavus pre-digested mucus (Fig.1). AIEC produced more H2S with R. gnavus-precultured mucus than with mucus alone. R. gnavus digestion of PGM released mucus monosaccharides (Fig.2A). AIEC strains grew differently with each monosaccharide constituent of R. gnavus pre-digested PGM. AIEC grew better than non-AIEC with each monosaccharide in aerobic conditions, but AIEC (B2) grew better than others in all monosaccharides in anaerobic conditions (Fig.2B). Amongst mucus-relevant sulfur substrates, L-cysteine was the only substrate to enhance E. coli H2S production by all AIEC and non-AIEC strains. All mucus-relevant sulfur substrates supported R. gnavus survival for 24 hrs, while H2S sustained R. gnavus for 48 hrs in minimum media. CONCLUSIONS AIEC grew better and produced more H2S with R. gnavus pre-digested mucus suggesting that co-existence of AIEC with R. gnavus mutually support each other and may explain why they are often observed together in active CD patients. In addition, enhanced AIEC growth with ileal vs. colonic mucus may explain why AIEC are frequently isolated from ileal CD patients. AIEC phylotype growth differences with mucus substrates may explain their persistence in the ileal mucosa. Figure 1 Figure 2

Action mechanism of the potential biocontrol agent Brevibacillus laterosporus SN19-1 against Xanthomonas oryzae pv. oryzae causing rice bacterial leaf blight.

The causal agent of rice bacterial leaf blight (BLB) is Xanthomonas oryzae pv. oryzae (Xoo), which causes serious damage to rice, leading to yield reduction or even crop failure. Brevibacillus laterosporus SN19-1 is a biocontrol strain obtained by long-term screening in our laboratory, which has a good antagonistic effect on a variety of plant pathogenic bacteria. In this study, we investigated the efficacy and bacterial inhibition of B. laterosporus SN19-1 against BLB to lay the theoretical foundation and research technology for the development of SN19-1 as a biopesticide of BLB. It was found that SN19-1 has the ability to fix nitrogen, detoxify organic phosphorus, and produce cellulase, protease, and siderophores, as well as IAA. In a greenhouse pot experiment, the control efficiency of SN19-1 against BLB was as high as 90.92%. Further investigation of the inhibitory mechanism of SN19-1 on Xoo found that the biofilm formation ability of Xoo was inhibited and the pathogenicity was weakened after the action of SN19-1 sterile supernatant on Xoo. The activities of enzymes related to respiration and the energy metabolism of Xoo were significantly inhibited, while the level of intracellular reactive oxygen species was greatly increased. Scanning electron microscopy observations showed folds on the surface of Xoo. A significant increase in cell membrane permeability and outer membrane permeability and a decrease in cell membrane fluidity resulted in the extravasation of intracellular substances and cell death. The results of this study highlight the role of B. laterosporus SN19-1 against the pathogen of BLB and help elucidate the underlying molecular mechanisms.

Proximal and Distal Bronchioles Contribute to the Pathogenesis of Non-Cystic Fibrosis Bronchiectasis.

Rationale: Non-cystic fibrosis bronchiectasis (NCFB) may originate in bronchiolar regions of the lung. Accordingly, there is a need to characterize the morphology and molecular characteristics of NCFB bronchioles. Objectives: Test the hypothesis that NCFB exhibits a major component of bronchiolar disease manifest by mucus plugging and ectasia. Methods: Morphologic criteria and region-specific epithelial gene expression, measured histologically and by RNA in situ hybridization and immunohistochemistry, identified proximal and distal bronchioles in excised NCFB lungs. RNA in situ hybridization and immunohistochemistry assessed bronchiolar mucus accumulation and mucin gene expression. CRISPR-Cas9-mediated IL-1R1 knockout in human bronchial epithelial cultures tested IL-1α and IL-1β contributions to mucin production. Spatial transcriptional profiling characterized NCFB distal bronchiolar gene expression. Measurements and Main Results: Bronchiolar perimeters and lumen areas per section area were increased in proximal, but not distal, bronchioles in NCFB versus control lungs, suggesting proximal bronchiolectasis. In NCFB, mucus plugging was observed in ectatic proximal bronchioles and associated nonectatic distal bronchioles in sections with disease. MUC5AC and MUC5B mucins were upregulated in NCFB proximal bronchioles, whereas MUC5B was selectively upregulated in distal bronchioles. Bronchiolar mucus plugs were populated by IL-1β-expressing macrophages. NCFB sterile sputum supernatants induced human bronchial epithelial MUC5B and MUC5AC expression that was >80% blocked by IL-1R1 ablation. Spatial transcriptional profiling identified upregulation of genes associated with secretory cells, hypoxia, interleukin pathways, and IL-1β-producing macrophages in mucus plugs and downregulation of epithelial ciliogenesis genes. Conclusions: NCFB exhibits distinctive proximal and distal bronchiolar disease. Both bronchiolar regions exhibit bronchiolar secretory cell features and mucus plugging but differ in mucin gene regulation and ectasia.

Transcriptomic and metabolomic analyses to study the key role by which Ralstonia insidiosa induces Listeria monocytogenes to form suspended aggregates.

Ralstonia insidiosa can survive in a wide range of aqueous environments, including food processing areas, and is harmful to humans. It can induce Listeria monocytogenes to form suspended aggregates, resulting from the co-aggregation of two bacteria, which allows for more persistent survival and increases the risk of L. monocytogenes contamination. In our study, different groups of aggregates were analyzed and compared using Illumina RNA sequencing technology. These included R. insidiosa under normal and barren nutrient conditions and in the presence or absence of L. monocytogenes as a way to screen for differentially expressed genes (DEGs) in the process of aggregate formation. In addition, sterile supernatants of R. insidiosa were analyzed under different nutrient conditions using metabolomics to investigate the effect of nutrient-poor conditions on metabolite production by R. insidiosa. We also undertook a combined analysis of transcriptome and metabolome data to further investigate the induction effect of R. insidiosa on L. monocytogenes in a barren environment. The results of the functional annotation analysis on the surface of DEGs and qPCR showed that under nutrient-poor conditions, the acdx, puuE, and acs genes of R. insidiosa were significantly upregulated in biosynthetic processes such as carbon metabolism, metabolic pathways, and biosynthesis of secondary metabolites, with Log2FC reaching 4.39, 3.96, and 3.95 respectively. In contrast, the Log2FC of cydA, cyoB, and rpsJ in oxidative phosphorylation and ribosomal pathways reached 3.74, 3.87, and 4.25, respectively. Thirty-one key components were identified while screening for differential metabolites, which mainly included amino acids and their metabolites, enriched to the pathways of biosynthesis of amino acids, phenylalanine metabolism, and methionine metabolism. Of these, aminomalonic acid and Proximicin B were the special components of R. insidiosa that were metabolized under nutrient-poor conditions.

Antifungal Activities of Bacillus mojavensis BQ-33 towards the Kiwifruit Black Spot Disease Caused by the Fungal Pathogen Didymella glomerata.

‘Hongyang’ kiwifruit (Actinidia chinensis, cultivar ‘Hongyang’) black spot disease is caused by the fungal pathogen Didymella glomerata, and is a serious disease, causing considerable losses to the kiwifruit industry during growth of the fruit. Hence, we aimed to identify a potential biocontrol agent against D. glomerata. In this study, bacterial isolates from the rhizosphere soil of kiwifruit were tested for their potential antifungal activity against selected fungal pathogens. Based on a phylogenetic tree constructed using sequences of 16S rDNA and the gyrA gene, BQ-33 with the best antifungal activity was identified as Bacillus mojavensis. We evaluated the antagonistic activity and inhibitory mechanism of BQ-33 against D. glomerata. Confrontation experiments showed that both BQ-33 suspension and the sterile supernatant (SS) produced by BQ-33 possessed excellent broad-spectrum antifungal activity. Furthermore, the SS damaged the cell membrane and cell wall of the mycelia, resulting in the leakage of a large quantity of small ions (Na+, K+), soluble proteins and nucleic acids. Chitinase and β-1,3-glucanase activities in SS increased in correlation with incubation time and remained at a high level for several days. An in vivo control efficacy assay indicated that 400 mL L−1 of SS completely inhibited kiwifruit black spot disease caused by D. glomerata. Therefore, BQ-33 is a potential biocontrol agent against kiwifruit black spot and plant diseases caused by other fungal pathogens. To our knowledge, this is the first report of the use of a rhizosphere microorganism as a biocontrol agent against kiwifruit black spot disease caused by D. glomerata.

Reduced alcohol preference and intake after fecal transplant in patients with alcohol use disorder is transmissible to germ-free mice

Alcohol use disorder is a major cause of morbidity, which requires newer treatment approaches. We previously showed in a randomized clinical trial that alcohol craving and consumption reduces after fecal transplantation. Here, to determine if this could be transmitted through microbial transfer, germ-free male C57BL/6 mice received stool or sterile supernatants collected from the trial participants pre-/post-fecal transplant. We found that mice colonized with post-fecal transplant stool but not supernatants reduced ethanol acceptance, intake and preference versus pre-fecal transplant colonized mice. Microbial taxa that were higher in post-fecal transplant humans were also associated with lower murine alcohol intake and preference. A majority of the differentially expressed genes (immune response, inflammation, oxidative stress response, and epithelial cell proliferation) occurred in the intestine rather than the liver and prefrontal cortex. These findings suggest a potential for therapeutically targeting gut microbiota and the microbial-intestinal interface to alter gut-liver-brain axis and reduce alcohol consumption in humans.

A New Perspective: Revealing the Algicidal Properties of Bacillus subtilis to Alexandrium pacificum from Bacterial Communities and Toxins.

Algicidal bacteria are important in the control of toxic dinoflagellate blooms, but studies on the environmental behavior of related algal toxins are still lacking. In this study, Bacillus subtilis S3 (S3) showed the highest algicidal activity against Alexandrium pacificum (Group IV) out of six Bacillus strains. When treated with 0.5% (v/v) S3 bacterial culture and sterile supernatant, the algicidal rates were 69.74% and 70.22% at 12 h, respectively, and algicidal substances secreted by S3 were considered the mechanism of algicidal effect. During the algicidal process, the rapid proliferation of Alteromonas sp. in the phycosphere of A. pacificum may have accelerated the algal death. Moreover, the algicidal development of S3 released large amounts of intracellular paralytic shellfish toxins (PSTs) into the water, as the extracellular PSTs increased by 187.88% and 231.47% at 12 h, compared with the treatment of bacterial culture and sterile supernatant at 0 h, respectively. Although the total amount of PSTs increased slightly, the total toxicity of the algal sample decreased as GTX1/4 was transformed by S3 into GTX2/3 and GTX5. These results more comprehensively reveal the complex relationship between algicidal bacteria and microalgae, providing a potential source of biological control for harmful algal blooms and toxins.

Strain-specific impacts of probiotics are a significant driver of gut microbiome development in very preterm infants

The development of the gut microbiome from birth plays important roles in short- and long-term health, but factors influencing preterm gut microbiome development are poorly understood. In the present study, we use metagenomic sequencing to analyse 1,431 longitudinal stool samples from 123 very preterm infants (<32 weeks’ gestation) who did not develop intestinal disease or sepsis over a study period of 10 years. During the study period, one cohort had no probiotic exposure whereas two cohorts were given different probiotic products: Infloran (Bifidobacterium bifidum and Lactobacillus acidophilus) or Labinic (B. bifidum, B. longum subsp. infantis and L. acidophilus). Mothers’ own milk, breast milk fortifier, antibiotics and probiotics were significantly associated with the gut microbiome, with probiotics being the most significant factor. Probiotics drove microbiome transition into different preterm gut community types (PGCTs), each enriched in a different Bifidobacterium sp. and significantly associated with increased postnatal age. Functional analyses identified stool metabolites associated with PGCTs and, in preterm-derived organoids, sterile faecal supernatants impacted intestinal, organoid monolayer, gene expression in a PGCT-specific manner. The present study identifies specific influencers of gut microbiome development in very preterm infants, some of which overlap with those impacting term infants. The results highlight the importance of strain-specific differences in probiotic products and their impact on host interactions in the preterm gut.

Histone Deacetylase Inhibition by Gut Microbe-Generated Short-Chain Fatty Acids Entrains Intestinal Epithelial Circadian Rhythms

The circadian clock orchestrates ∼24-hour oscillations of gastrointestinal epithelial structure and function that drive diurnal rhythms in gut microbiota. Here, we use experimental and computational approaches in intestinal organoids to reveal reciprocal effects of gut microbial metabolites on epithelial timekeeping by an epigenetic mechanism. We cultured enteroids in media supplemented with sterile supernatants from the altered Schaedler Flora (ASF), a defined murine microbiota. Circadian oscillations of bioluminescent PER2 and Bmal1 were measured in the presence or absence of individual ASF supernatants. Separately, we applied machine learning to ASF metabolomics to identify phase-shifting metabolites. Sterile filtrates from 3 of 7 ASF species (ASF360 Lactobacillus intestinalis, ASF361 Ligilactobacillus murinus, and ASF502 Clostridium species) induced minimal alterations in circadian rhythms, whereas filtrates from 4 ASF species (ASF356 Clostridium species, ASF492 Eubacterium plexicaudatum, ASF500 Pseudoflavonifactor species, and ASF519 Parabacteroides goldsteinii) induced profound, concentration-dependent phase shifts. Random forest classification identified short-chain fatty acid (SCFA) (butyrate, propionate, acetate, and isovalerate) production as a discriminating feature of ASF "shifters." Experiments with SCFAs confirmed machine learning predictions, with a median phase shift of 6.2 hours in murine enteroids. Pharmacologic or botanical histone deacetylase (HDAC) inhibitors yielded similar findings. Further, mithramycin A, an inhibitor of HDAC inhibition, reduced SCFA-induced phase shifts by 20% (P < .05) and conditional knockout of HDAC3 in enteroids abrogated butyrate effects on Per2 expression. Key findings were reproducible in human Bmal1-luciferase enteroids, colonoids, and Per2-luciferase Caco-2 cells. Gut microbe-generated SCFAs entrain intestinal epithelial circadian rhythms by an HDACi-dependent mechanism, with critical implications for understanding microbial and circadian network regulation of intestinal epithelial homeostasis.

Cutibacterium acnes biofilm forming clinical isolates modify the formation and structure of Staphylococcus aureus biofilms, increasing their susceptibility to antibiotics

Cutibacterium acnes (formally Propionibacterium acnes) is frequently identified within surgical device related infections. It is often co-isolated from infection sites with other opportunistic pathogens. Recent studies have demonstrated that C. acnes is able to form biofilms and when co-cultured with Staphylococcus spp. both inhibitory and stimulatory effects have been reported across several studies. Here, we investigated the biofilm-forming ability of 100 clinical C. acnes isolates from various infection sites in human patients, both deep tissue and superficial, followed by an investigation of how the supernatants of C. acnes cultures influenced the attachment and maturation of Staphylococcusaureus NCTC 6571 biofilms. All of the C. acnes isolates were able to form biofilms in vitro, although biofilm biomass varied between isolates. Nineteen isolates were weakly adherent, 33 were moderately adherent and the majority (48) showed strong adherence. The presence of C. acnes sterile supernatants reduced the biomass of S. aureus cultures, with a > 90% reduction observed in the presence of several of the C. acnes isolates. We observed that this decrease was not due to C. acnes affecting S. aureus viability, nor due to the presence of propionic acid. Biofilm maturation was however delayed over a 24-h period as was biofilm surface structure, although initial (up to 8 h) surface attachment was not affected. We hypothesis that this defective biofilm maturation is the cause of the observed biomass decrease. In turn, these altered biofilms showed a greater susceptibility to antibiotic treatments. In contrast the presence of C. acnes supernatant in planktonic (defined as a free moving, non-surface attached population within the liquid column) S. aureus cultures increased antibiotic tolerance, via a currently undefined mechanism. This study suggests that complex interactions between C. acnes and other opportunistic pathogens are likely to exist during colonisation and infection events. Further investigation of these interactions may lead to increased treatment options and a better prognosis for patients.

Subacute ruminal acidosis phenotypes in periparturient dairy cows differ in ruminal and salivary bacteria and in the in vitro fermentative activity of their ruminal microbiota

Both ruminal microbial structure and functionality might play a role in inter-individual variation in susceptibility for subacute rumen acidosis (SARA) observed in dairy cows. The aims of this study were to determine whether differences between cows with distinct SARA susceptibility were reflected in distinct (1) ruminal microbial communities, (2) salivary bacterial communities, and (3) fermentative capacity of ruminal microbiota assessed in vitro. To test this hypothesis, rumen samples were collected via an esophageal tube on 21 d postpartum from 38 multiparous Holstein cows, which were classified into 4 groups differing in median and mean time of reticular pH below 6 as well as area under the curve of pH below 6.0. During the 21 d postpartum, all cows within a group fulfilled following criteria: susceptible (S, n = 10; mean or median ≥180 min/d), moderately susceptible (MS, n = 7; 60 min/d < mean time of pH below 6 < 180 min/d, and median time of pH below 6 <180 min/d), moderately unsusceptible (MU, n = 11; 10 min/d < mean < 60 min/d, and median time of pH below 6 ≤30 min/d), or unsusceptible (U, n = 10; median = 0 min/d, and mean <10 min/d). Groups did not differ in total daily dry matter intake nor in total, roughage, or concentrate intake during daily 6-h time intervals. Rumen bacterial α-diversity did not differ among groups, but β-diversity varied and bacterial 16S rRNA gene copy numbers were lower in S compared with U cows. The relative abundance of genera Streptococcus, Sharpea, Prevotellaceae_YAB2003, Succinivibrionaceae_UCG-001, Ruminococcus, and Ruminococcaceae_UCG-001 were higher in S compared with U cows. In contrast, Lachnospiraceae_ND3007 and Oscillospiraceae_V9D2013 were more abundant in U cows. Although pH-associated, inter-animal differences were also observed in the salivary bacteria, common differences in ruminal and salivary bacterial genera were limited. The functionality of the rumen microbiota was evaluated in vitro through exposure of the microbial inoculum of S and U cows to an anaerobic buffer at pH 5.8 and 6.8, in the presence of sterile supernatant of their own and of dry cows' rumen fluid (2 × 2 design). Generally, the S inoculum produced more volatile fatty acids, except at low pH with dry cows' supernatant, where volatile fatty acid production was completely impaired and lactate accumulation was highest. Compared with the microbes of U cows, microbes of S cows showed less fermentative activity in situations with 2 stress factors (low pH and an unfamiliar environment, i.e., rumen fluid supernatant of dry cows).

Use of bacterial culture supernatants as anti-biofilm agents against Pseudomonas aeruginosa and Klebsiella pneumoniae.

Pseudomonas aeruginosa and Klebsiella pneumoniae are the most pervasive and challenging agents of bacterial nosocomial infections. Previous studies indicated that the microbial biofilms formed by these bacteria may play important roles in their pathogenesis and resistance to phagocytosis and antibiotics. The aim of this study was to explore the anti-biofilm activity of culture supernatants of Salmonella enterica subsp. enteric serovar Typhimurium SL1344 and P. aeruginosa PA01 against biofilms formed by P. aeruginosa PA01 and K. pneumoniae KR3167, respectively. Biofilm formation was quantified by crystal violet staining. A modified method was applied to separate planktonic and biofilm-forming cells. The viable cells in the planktonic and biofilm phases were quantitated by viable plate count. Dual-species interactions between P. aeruginosa PAO1 and Salmonella enterica subsp. enterica serovar Typhimurium SL1344 were investigated using different cell density ratios. Biofilm formation of P. aeruginosa PA01 was significantly inhibited by the heat resistant components from the culture supernatants of Salmonella enterica subsp. enterica serovar Typhimurium. Biofilm formed by K. pneumoniae KR3167 was also inhibited by the culture supernatants of P. aeruginosa PA01. The supernatants obtained from planktonic cell caused greater biofilm reduction than those extracted from biofilm-forming cells. This study is the first to report that sterile crude supernatants extracted from the cultures of Salmonella enterica and P. aeruginosa significantly inhibited biofilm formation of P. aeruginosa and K. pneumoniae, respectively. The active agents in the culture supernatants responsible for biofilm inhibition have not been determined yet. The culture supernatants of Salmonella enterica and P. aeruginosa should be further studied for their therapeutic potential to reduce biofilm formation produced by bacteria causing nosocomial infections.

High throughput screening of technological and biopreservation traits of a large set of wild lactic acid bacteria from Brazilian artisanal cheeses

This study aimed to evaluate technological (acidification, proteolysis, lipolysis, resistance to low pH, NaCl, and bile salts) and biopreservation (antimicrobial activity against foodborne pathogens) features of 1002 LAB by high throughput screening (HTS) methods. The LAB was isolated from 11 types of Brazilian artisanal cheeses (BAC) marketed in the main 5 producing regions. Remarkable intra-species variability in acidification rates have been found, which was most pronounced between isolates from Mina's artisanal cheeses, Caipira and Coalho cheeses. Lacticaseibacillus paracasei and Levilactobacillus brevis showed the fastest acidification rate; however, all isolates showed slower acidification rates than a lactococcal control strain (4.3 × lower). When testing inhibitory effects, > 75% of LAB isolates could inhibit the growth of Staphylococcus aureus ATCC 19095 and Listeria monocytogenes ATCC 7644. Two of these isolates, identified as Lactiplantibacillus plantarum and Lentilactobacillus buchneri, the sterile and neutral supernatants alone, were sufficient to inhibit L. monocytogenes growth. Principal component analysis (PCA) allowed the identification of functional groups based on proteolytic and lipolytic activity, osmotic stress resistance, and inhibition of L. monocytogenes. The type of cheese the isolates were recovered from influenced properties such as anti-listerial compounds and lipolytic enzyme production. The use of HTS and multivariate statistics allowed insights into a diverse set of LAB technological and biopreservation properties. These findings allow a profound knowledge of the heterogeneity of a large set of isolates, which can be further used to design starter cultures with varied and combined properties, such as biopreservation and technological features. Besides that, HTS makes it possible to analyze a vast panel of LAB strains, reducing costs and time within laboratory analysis, while avoiding the loss of information once all LAB are tested at the same time (differently from the traditional labor-intensive approach, in which a few numbers of strains is tested per time).

Gram Negatif Bakterilerin ve Steril Süpernatantlarının Candida albicans Biyofilmi Üzerine Etkilerinin Karşılaştırılması

Objective: Polymicrobial biofilms consisting of a combination of various bacteria and/or fungi are generally much more resistant than monomicrobial biofilms formed by these species alone. In this study, it was aimed to investigate how Candida albicans biofilms were affected in the presence of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii or Pseudomonas aeruginosa or supernatants of these bacteria. Method: C. albicans with Gram-negative polymicrobial biofilms were formed on sterile microplates by using control strains of C. albicans SC 5314, E. coli ATCC 25922, K. pneumoniae ATCC 700603, A. baumannii ATCC 19606 and P. aeruginosa PA01. The number of C. albicans in biofilms was determined in the presence of both Gram- negative bacteria and sterile supernatants. Results: According to our results, all Gram negative bacteria displayed an antagonist effect against C. albicans in the biofilm and a three log decrease was observed compared to the control. Sterile supernatants were shown to have an inhibitory effect on the C. albicans biofilms and reduce the number of yeasts by at least one log. MTT assay and fluorescence microscopy images also confirmed the results. Conclusion: In C. albicans-Gram-negative polymicrobial biofilms that can occur in many infections, bacteria affected C. albicans biofilm cells as antagonist agents, both with their cells and sterile cell-free supernatants

Gut microbiota derived metabolites contribute to intestinal barrier maturation at the suckling-to-weaning transition

ABSTRACT In suckling mammals, the onset of solid food ingestion is coincident with the maturation of the gut barrier. This ontogenic process is driven by the colonization of the intestine by the microbiota. However, the mechanisms underlying the microbial regulation of the intestinal development in early life are not fully understood. Here, we studied the co-maturation of the microbiota (composition and metabolic activity) and of the gut barrier at the suckling-to-weaning transition by using a combination of experiments in vivo (suckling rabbit model), ex vivo (Ussing chambers) and in vitro (epithelial cell lines and organoids). The microbiota composition, its metabolic activity, para-cellular epithelial permeability and the gene expression of key components of the gut barrier shifted sharply at the onset of solid food ingestion in vivo, despite milk was still predominant in the diet at that time. We found that cecal content sterile supernatant (i.e. containing a mixture of metabolites) obtained after the onset of solid food ingestion accelerated the formation of the epithelial barrier in Caco-2 cells in vitro and our results suggested that these effects were driven by the bacterial metabolite butyrate. Moreover, the treatment of organoids with cecal content sterile supernatant partially replicated in vitro the effects of solid food ingestion on the epithelial barrier in vivo. Altogether, our results show that the metabolites produced by the microbiota at the onset of solid food ingestion contribute to the maturation of the gut barrier at the suckling-to-weaning transition. Targeting the gut microbiota metabolic activity during this key developmental window might therefore be a promising strategy to promote intestinal homeostasis.

Prebiotics can change immunomodulatory properties of probiotics.

Beneficial effects of probiotics and prebiotics are mainly related to modulation of compositions and activities of gut microbiota as well as manipulation of immunological reactivity in autoimmune diseases. In the present study, we examined whether metabolic products from different strains of Lactobacillus brevis cultured with different prebiotics have similar immunomodulating properties on immune cells under normal and inflammatory conditions, using mouse model of collagen-induced arthritis (CIA). Two strains of Lactobacillus brevis (3448 and 8429) were cultured with four different prebiotics, such as xylooligosaccharides, inulin, pectin, and chitosan. Sterile supernatants containing different metabolic products have been used for direct treatment of cell cultures prepared from CII-immunized mice and non-immunized (control mice). Our results showed that metabolic products from XOS decreased levels of IFN-γ, IL-6, IL-17, and TNF-α in both cultures from immunized and non-immunized mice. In contrast, metabolic products from inulin, pectin, and chitosan increased concentrations of these cytokines with highest values for pectin. Neither of investigated prebiotics influenced the secretion of IL-10. In addition, we found changes in the percentage of macrophages, which were different for the tested prebiotics. Also, metabolic products from pectin and chitosan caused loss of T-cells (CD3+) and increased percentages of CD4+CD25+ regulatory T cells and CD8+CD279+ anergic T cells. Hence, our data indicate that immunomodulating properties of probiotics are strain-specific and prebiotic-dependent.

Neuroinflammation in Murine Cirrhosis Is Dependent on the Gut Microbiome and Is Attenuated by Fecal Transplant.

Cirrhosis and hepatic encephalopathy (HE) is associated with an altered gut-liver-brain axis. Fecal microbial transplant (FMT) after antibiotics improves outcomes in HE, but the impact on brain function is unclear. The aim of this study is to determine the effect of colonization using human donors in germ-free (GF) mice on the gut-liver-brain axis. GF and conventional mice were made cirrhotic using carbon tetrachloride and compared with controls in GF and conventional state. Additional GF mice were colonized with stool from controls (Ctrl-Hum) and patients with cirrhosis (Cirr-Hum). Stools from patients with HE cirrhosis after antibiotics were pooled (pre-FMT). Stools from the same patients 15 days after FMT from a healthy donor were also pooled (post-FMT). Sterile supernatants were created from pre-FMT and post-FMT samples. GF mice were colonized using stools/sterile supernatants. For all mice, frontal cortex, liver, and small/large intestines were collected. Cortical inflammation, synaptic plasticity and gamma-aminobutyric acid (GABA) signaling, and liver inflammation and intestinal 16s ribosomal RNA microbiota sequencing were performed. Conventional cirrhotic mice had higher degrees of neuroinflammation, microglial/glial activation, GABA signaling, and intestinal dysbiosis compared with other groups. Cirr-Hum mice had greater neuroinflammation, microglial/glial activation, and GABA signaling and lower synaptic plasticity compared with Ctrl-Hum mice. This was associated with greater dysbiosis but no change in liver histology. Pre-FMT material colonization was associated with neuroinflammation and microglial activation and dysbiosis, which was reduced significantly with post-FMT samples. Sterile pre-FMT and post-FMT supernatants did not affect brain parameters. Liver inflammation was unaffected. Conclusion: Fecal microbial colonization from patients with cirrhosis results in higher degrees of neuroinflammation and activation of GABAergic and neuronal activation in mice regardless of cirrhosis compared with those from healthy humans. Reduction in neuroinflammation by using samples from post-FMT patients to colonize GF mice shows a direct effect of fecal microbiota independent of active liver inflammation or injury.