Live Bacteria Research Articles

Portable ATP bioluminescence sensor with high specificity for live Escherichia coli O157:H7 strain synergistically enhanced by orientated phage-modified stir bar extraction and bio-proliferation

Live foodborne pathogens proliferate rapidly and do great harm to human health, which requires appropriate methods to supervise. In this work, a portable adenosine triphosphate (ATP) bioluminescence sensor with high specificity for live E. coli O157:H7 strain synergistically enhanced by orientated phage-modified stir bar extraction and bio-proliferation was developed. In brief, the selected phages were directionally immobilized on the poly(diallyldimethylammonium chloride)-modified gold stir bar as the bioreceptor. Following the simple stir bar absorptive extraction and bio-proliferation in the Luria-Bertani medium, the number of captured E. coli O157:H7 exploded. Finally, it was quantified by a portable ATP bioluminescence sensor. Benefitting from the high specificity of phage and simple signal dual-amplification strategy, the proposed biosensor achieved the recognition of live bacteria at strain level with superior sensitivity. Also, the portable signal readout made it suitable for on-site detection. Under optimal conditions, this bioassay provided a detectable range of 102–107 CFU mL−1 with a low detection limit of 30 CFU mL−1 within 30 min. The detection results for real samples demonstrated that there were no differences between the assay and the plate counting method, while the detection time was largely shortened. Furthermore, the assay gives a novel path for the point-of-care test (POCT) of live E. coli strain, which is promising to be extended to other virulent strains measurement with corresponding phages.

Manipulating the microbiome to enhance oral tolerance in food allergy

Loss of oral tolerance (OT) to food antigens results in food allergies. One component of achieving OT is the symbiotic microorganisms living in the gut (microbiota). The composition of the microbiota can drive either pro-tolerogenic or pro-inflammatory responses against dietary antigens though interactions with the local immune cells within the gut. Products from bacterial fermentation, such as butyrate, are one of the main communication molecules involved in this interaction, however, this is released by a subset of bacterial species. Thus, strategies to specifically expand these bacteria with protolerogenic properties have been explored to complement oral immunotherapy in food allergy. These approaches either provide digestible biomolecules to induce beneficial bacteria species (prebiotics) or the direct administration of live bacteria species (probiotics). While this combined therapy has shown positive outcomes in clinical trials for cow’s milk allergy, more research is needed to determine if this therapy can be extended to other food allergens.

Helicobacter pylori Infection Elicits Type I Interferon Response in Human Monocytes via Toll-Like Receptor 8 Signaling.

Helicobacter pylori colonization and persistence could precede gastric adenocarcinoma. Elucidating immune recognition strategies of H. pylori is therefore imperative to curb chronic persistence in the human host. Toll-like receptor 7 (TLR7) and TLR8 are widely known as viral single-stranded RNA (ssRNA) sensors yet less studied in the bacteria context. Here, we investigated the involvement of these receptors in the immunity to H. pylori. Human THP-1 monocytic cells were infected with H. pylori, and the expression levels of human Toll-like receptors (TLRs) were examined. The roles of TLR7 and TLR8 in response to H. pylori infection were further investigated using receptor antagonists. Among all TLR transcripts examined, TLR8 exhibited the most prominent upregulation, followed by TLR7 in the THP-1 cells infected with H. pylori J99 or SS1 strains. H. pylori infection-mediated IFN-α and IFN-β transactivation was significantly abrogated by the TLR7/8 (but not TLR7) antagonist. Additionally, TLR7/8 antagonist treatment reduced H. pylori infection-mediated phosphorylation of interferon regulatory factor 7 (IRF7). Our study suggests a novel role of TLR8 signaling in host immunity against H. pylori through sensing live bacteria to elicit the production of type I interferon.

Advances of Rumen Functional Bacteria and the Application of Micro-Encapsulation Fermentation Technology in Ruminants: A Review

Rumen functional bacteria are crucial for the homeostasis of rumen fermentation and micro-ecology. Cellulolytic bacteria, amylolytic bacteria, protein- and fat-degrading bacteria, lactic acid-producing bacteria, lactic acid-consuming bacteria, methanogens, and others can all be found in the rumen flora and help the host and other microorganisms convert feed into energy. For instance, Ruminococcus flavefaciens, Ruminococcus albus, and Fibrobacter succinogenes are the three most prevalent fiber-degrading bacteria. The digestion and metabolism of various nutrients and the absorption in rumen epithelium can greatly enhance host defense mechanisms and health production in ruminants. However, directly feeding live bacteria is prone to negative environmental effects. Therefore, the micro-encapsulation of film-forming and acid-resistant wall materials can become a great means of encapsulating naked bacteria into tiny particles. It can maintain the activity of functional flora, boost the function of the intestinal barrier, and improve its capacity for colonization on the surface of the rumen and colon mucosa. Therefore, the present review evaluates the latent progress of main functional bacteria and the applied techniques of micro-encapsulation in the rumen, in order to provide more references for the development and application of rumen-functional bacteria.

Assessing the Neurotoxicity of a Sub-Optimal Dose of Rotenone in Zebrafish (Danio rerio) and the Possible Neuroactive Potential of Valproic Acid, Combination of Levodopa and Carbidopa, and Lactic Acid Bacteria Strains.

Parkinson’s disease (PD) is an enigmatic neurodegenerative disorder that is currently the subject of extensive research approaches aiming at deepening the understanding of its etiopathophysiology. Recent data suggest that distinct compounds used either as anticonvulsants or agents usually used as dopaminergic agonists or supplements consisting of live active lactic acid bacteria strains might alleviate and improve PD-related phenotypes. This is why we aimed to elucidate how the administration of rotenone (ROT) disrupts homeostasis and the possible neuroactive potential of valproic acid (VPA), antiparkinsonian agents (levodopa and carbidopa – LEV+CARB), and a mixture of six Lactobacillus and three Bifidobacterium species (PROBIO) might re-establish the optimal internal parameters. ROT causes significant changes in the central nervous system (CNS), notably reduced neurogenesis and angiogenesis, by triggering apoptosis, reflected by the increased expression of PARKIN and PINK1 gene(s), low brain dopamine (DA) levels, and as opposed to LRRK2 and SNCA compared with healthy zebrafish. VPA, LEV/CARB, and PROBIO sustain neurogenesis and angiogenesis, manifesting a neuroprotective role in diminishing the effect of ROT in zebrafish. Interestingly, none of the tested compounds influenced oxidative stress (OS), as reflected by the level of malondialdehyde (MDA) level and superoxide dismutase (SOD) enzymatic activity revealed in non-ROT-exposed zebrafish. Overall, the selected concentrations were enough to trigger particular behavioral patterns as reflected by our parameters of interest (swimming distance (mm), velocity (mm/s), and freezing episodes (s)), but sequential testing is mandatory to decipher whether they exert an inhibitory role following ROT exposure. In this way, we further offer data into how ROT may trigger a PD-related phenotype and the possible beneficial role of VPA, LEV+CARB, and PROBIO in re-establishing homeostasis in Danio rerio.

Christensenella regulated by Huang-Qi-Ling-Hua-San is a key factor by which to improve type 2 diabetes.

There is a lot of evidence that oral hypoglycemic drugs work by affecting gut microbes, but the key strains responsible for this effect are not well known. Huang-Qi-Ling-Hua-San (HQLHS), composed of Astragalus Membranaceus, Ganoderma lucidum, Inonotus obliquus, and Momordica charantia L., is a specially designed Chinese medicine formula to treat type 2 diabetes (T2D). In this study, a mouse model of T2D induced by high-fat diet and streptozotocin was used to explore the mechanism of HQLHS in improving hyperglycemia and hyperlipidemia through multiple rounds of animal experiments, such as HQLHS feeding, fecal microbiota transplantation (FMT), and live bacteria feeding, so as to explore the potential target intestinal flora in its hypoglycemic effect. Results show that such specific taxa as Bifidobacterium, Turicibacter, Alistipes, Romboutsia, and Christensenella were identified to be preferably enriched by HQLHS and then assumed to be the target microbes. Herein, FMT was used to test if the upregulated beneficial bacteria by HQLHS play a therapeutic role. The strain Christensenella minuta DSM 22607 and the strain Christensenella timonensis DSM 102800 were selected to test the beneficial effect of Christensenella taxa on T2D. Diabetic animals supplemented with these strains showed the improvement in blood glucose and lipid metabolism, the promotion of GLP-1 secretion, the increase in antioxidant capacity, the inhibition of hepatic gluconeogenesis, the suppression of intestinal glucose absorption, the enhancement of intestinal barrier, reduced LPS-induced inflammation, and the reduction of branched amino acids (BCAAs) content in the liver. Overall, these data demonstrate that Christensenella plays a beneficial role in T2D and is a target for the action of HQLHS therapy.

Constraints Faced By Farmers in Use of Private Bio-Fertilizers: A Way Forward

The present study was conducted at Navsari district of South Gujarat. 120 respondents were selected for the study. Ex-post facto research design was used for the study. The bio-fertilizer refers to a formulation containing live bacteria that improve soil fertility by fixing atmospheric nitrogen, solubilizing phosphorus and other nutrients, and boosting plant development by releasing growth hormones. Because the concept is not new, the numerous benefits of bio-fertilizer led to its widespread application in sustainable agriculture. In this context private bio-fertilizers refers to the bio-fertilizers are the bio-fertilizers manufactured by private manufacturers or companies. The result of the study revealed that, Lack of technical knowledge about private bio-fertilizers was the major constraint followed, Non-availability of private bio-fertilizer locally at times when needed, lack of awareness about benefits of private bio-fertilizers etc. whereas, private bio-fertilizers should be available in time, bio-fertilizer usage can be increased if they are provided free of cost etc. are the suggestions given by the respondents.

SpyPhage: A Cell-Free TXTL Platform for Rapid Engineering of Targeted Phage Therapies.

The past decade has seen the emergence of multidrug resistant pathogens as a leading cause of death worldwide, reigniting interest in the field of phage therapy. Modern advances in the genetic engineering of bacteriophages have enabled several useful results including host range alterations, constitutive lytic growth, and control over phage replication. However, the slow licensing process of genetically modified organisms clearly inhibits the rapid therapeutic application of novel engineered variants necessary to fight mutant pathogens that emerge throughout the course of a pandemic. As a solution to this problem, we propose the SpyPhage system where a "scaffold" bacteriophage is engineered to incorporate a SpyTag moiety on its capsid head to enable rapid postsynthetic modification of their surfaces with SpyCatcher-fused therapeutic proteins. As a proof of concept, through CRISPR/Cas-facilitated phage engineering and whole genome assembly, we targeted a SpyTag capsid fusion to K1F, a phage targeting the pathogenic strain Escherichia coli K1. We demonstrate for the first time the cell-free assembly and decoration of the phage surface with two alternative fusion proteins, SpyCatcher-mCherry-EGF and SpyCatcher-mCherry-Rck, both of which facilitate the endocytotic uptake of the phages by a urinary bladder epithelial cell line. Overall, our work presents a cell-free phage production pipeline for the generation of multiple phenotypically distinct phages with a single underlying "scaffold" genotype. These phages could become the basis of next-generation phage therapies where the knowledge-based engineering of numerous phage variants would be quickly achievable without the use of live bacteria or the need to repeatedly license novel genetic alterations.

Modulation of the extracellular matrix by Streptococcus gallolyticus subsp. gallolyticus and importance in cell proliferation.

Streptococcus gallolyticus subspecies gallolyticus (Sgg) has a strong clinical association with colorectal cancer (CRC) and actively promotes the development of colon tumors. Previous work showed that this organism stimulates CRC cells proliferation and tumor growth. However, the molecular mechanisms underlying these activities are not well understood. Here, we found that Sgg upregulates the expression of several type of collagens in HT29 and HCT116 cells, with type VI collagen (ColVI) being the highest upregulated type. Knockdown of ColVI abolished the ability of Sgg to induce cell proliferation and reduced the adherence of Sgg to CRC cells. The extracellular matrix (ECM) is an important regulator of cell proliferation. Therefore, we further examined the role of decellularized matrix (dc-matrix), which is free of live bacteria or cells, in Sgg-induced cell proliferation. Dc-matrix prepared from Sgg-treated cells showed a significantly higher pro-proliferative activity than that from untreated cells or cells treated with control bacteria. On the other hand, dc-matrix from Sgg-treated ColVI knockdown cells showed no difference in the capacity to support cell proliferation compared to that from untreated ColVI knockdown cells, suggesting that the ECM by itself is a mediator of Sgg-induced cell proliferation. Furthermore, Sgg treatment of CRC cells but not ColVI knockdown CRC cells resulted in significantly larger tumors in vivo, suggesting that ColVI is important for Sgg to promote tumor growth in vivo. These results highlight a dynamic bidirectional interplay between Sgg and the ECM, where Sgg upregulates collagen expression. The Sgg-modified ECM in turn affects the ability of Sgg to adhere to host cells and more importantly, acts as a mediator for Sgg-induced CRC cell proliferation. Taken together, our results reveal a novel mechanism in which Sgg stimulates CRC proliferation through modulation of the ECM.

Psychobiotics and Dietary Supplementation in the Clinical Practice: A Mini Review

Mental health defined as a state of well-being in which the individual realizes his or her own abilities, can cope with the normal stresses of life, can work productively and fruitfully, and is able to make meaningful contribution to his or her community‟ reflects the individual ability to adapt to changing environment through thoughts, emotions and behaviors. Recently, the prevalence of psychiatric diseases is an emerging health issue that significantly impact on the quality of life and productivity of societies worldwide. Despite recent research advancements, the effectiveness off the curative approaches for the mental illness are still limited to pharmacologic treatments and/or psychotherapy, thereby indicating the need of development of alternative strategies. Growing number of research efforts into the better understanding of mental health determinants, prompted scientific interests into the use of dietary interventions, as potential approach to modify the mental state and improve wellbeing. A growing body of evidence to demonstrate the importance of diet, in particular intake of plant foods, in achieving optimal nutritional and health status, which has been attributed to plant-derived bioactives that consumed as whole food or dietary supplement may provide benefits for mental health, therefore providing an alternative for common pharmacotherapies in the psychiatry practice applied to treat metal conditions, including anxiety, depression, ADHD or schizophrenia. In addition, the discovery of a bidirectional communication between brain and gut microbiome, followed by the studies linking thee gut microbiota composition with behavior and mood, support use of microbial-derived metabolites in the form of live probiotic bacteria (probiotics), as well as their metabolites (post-biotics) may help in stress management and prevent anxiety-/depression-like symptoms in high-risk populations.

Postbiotics: From emerging concept to application

The microbiome innovation has resulted in an umbrella term, postbiotics, which refers to non-viable microbial cells, metabolic byproducts and their microbial components released after lysis. Postbiotics, modulate immune response, gene expression, inhibit pathogen binding, maintain intestinal barriers, help in controlling carcinogenesis and pathogen infections. Postbiotics have antimicrobial, antioxidant, and immunomodulatory properties with favorable physiological, immunological, neuro-hormonal, regulatory and metabolic reactions. Consumption of postbiotics relieves symptoms of various diseases and viral infections such as SARS-CoV-2. Postbiotics can act as alternatives for pre-probiotic specially in immunosuppressed patients, children and premature neonates. Postbiotics are used to preserve and enhance nutritional properties of food, elimination of biofilms and skin conditioning in cosmetics. Postbiotics have numerous advantages over live bacteria with no risk of bacterial translocation from the gut to blood, acquisition of antibiotic resistance genes. The process of extraction, standardization, transport, and storage of postbiotic is more natural. Bioengineering techniques such as fermentation technology, high pressure etc., may be used for the synthesis of different postbiotics. Safety assessment and quality assurance of postbiotic is important as they may induce stomach discomfort, sepsis and/or toxic shock. Postbiotics are still in their infancy compared to pre- and pro- biotics but future research in this field may contribute to improved physiological functions and host health. The current review comprehensively summarizes new frontiers of research in postbiotics.

PSVII-17 Feeding Behavior of Beef Feedlot Yearling Steers Offered Finishing Diets with Direct fed Microbial Mixtures

Abstract The effects of dietary inclusion of live bacteria mixtures on feedlot beef cattle feeding behavior were evaluated. Crossbred-Angus yearling steers (n = 192; initial BW = 409 kg ± 8 kg) were blocked by body weight (BW) and randomly assigned into 48 pens (4 steers/pen; 16 pens/treatment) following a randomized complete block design. A steam-flaked corn-based fishing diet was offered ad libitum once daily containing the following treatments: 1) Control, in which no direct fed microbial (DFM) was offered (lactose as carrier only); 2 and 3) Probiotic mixtures at distinct concentrations [Mixture A and B, at 2g/animal-daily (lactose used as carrier)]. Orts DM were quantified daily and subtracted from total dietary DM offered to calculate DM intake. Feeding behavior activities (continuous 24 h) were visually assessed (d 106 and 113) by trained personnel, where time spent on each activity was recorded every 5 min. Data were analyzed using the GLIMMIX procedures of SAS and pen was considered the experimental unit. No treatment × period interactions (P ≥ 0.21) were observed. Main feeding behavior variables consisting of time spent ruminating, chewing, eating, and drinking in minutes per day were not affected (P ≥ 0.17) by treatments, while steers in general ruminated, ate, chewed, and were more active (P ≤ 0.01) during the second behavioral assessment, regardless of treatments. Rates of intake in minutes per kg of DM, OM, fiber and digestible DM, OM, and fiber were not affected (P ≥ 0.12) by treatments; however more time spent (P < 0.01) ruminating, eating, and chewing in minutes per kg were observed on d113 compared to d106 regardless of treatments. Drinking time (min/d) and drinking rates (min/kg) were not affected (P ≥ 0.81) by period. The DFM mixtures did not negatively affect feeding behavior of yearling steers consuming steam-flaked corn-based finishing diets.

339 Growth Performance and Carcass Characteristics of Feedlot Beef Yearling Steers Offered Finishing Diets with Direct fed Microbial Mixtures

Abstract The effects of dietary inclusion of live bacteria on feedlot beef cattle growth performance and carcass characteristics were evaluated. Crossbred-Angus yearling steers (n = 192; initial BW = 409 kg ± 8 kg) were blocked by body weight (BW) and randomly assigned into 48 pens (4 steers/pen; 16 pens/treatment) following a randomized complete block design. A steam-flaked corn-based fishing diet was offered ad libitum once daily containing the following treatments: 1) Control, in which no direct fed microbial (DFM) was offered (lactose as carrier only); 2 and 3) Probiotic mixtures at distinct concentrations [Mixture A and B, at 2g/animal-daily (lactose used as carrier)]. Orts DM were quantified daily and subtracted from total dietary DM offered to calculate DM intake. Two-day consecutive unshrunk BW were recorded before feeding on d 0, 30, 60, 90, 121, and 153 (prior to shipment to a federally inspected slaughter facility). Data were analyzed using the GLIMMIX procedure of SAS and pen was considered the experimental unit, in which F-test protected pre-planned contrasts comparing control versus DFM mixture-A and control versus DFM mixture-B were used. Steers offered mixture-A increased carcass-adjusted ADG (P = 0.03) by 6.7%, gain efficiency (P < 0.01) by 6%, tended to increase carcass-adjusted final BW (P = 0.07) by 15kg and hot carcass weight (P = 0.07) by 10kg. The overall (d 0 to end) DM intake (P = 0.36) was not affected by treatment; however, a subtle (1.2%) decrease (P < 0.01) during the initial 30 days for steers offered DFM mixture-B was observed. Carcass variables (dressing percentage, 12th rib fat, longissimus muscle area, marbling, yield grade, and liver scores) were not affected (P ≥ 0.13) by treatments. Growth performance was improved with DFM mixture-A which seemed to positively affect carcass weight without inducing deleterious effects on other carcass characteristics.

PSIX-6 Ruminal Tissue Morphology of Beef Yearling Steers Offered Finishing Diets with Direct fed Microbial Mixtures

Abstract The effects of the dietary inclusion of live bacteria mixtures on feedlot beef cattle ruminal papillae morphology were evaluated. Crossbred-Angus yearling steers (n = 192; initial BW = 409 kg ± 8 kg) were blocked by body weight (BW) and randomly assigned into 48 pens (4 steers/pen; 16 pens/treatment) following a randomized complete block design. A steam-flaked corn-based fishing diet was offered ad libitum once daily during 153 d containing the following treatments: 1) Control, in which no direct-fed microbial (DFM) was offered (lactose as carrier only); 2 and 3) Probiotic mixtures at distinct concentrations [Mixture A and B, at 2g/animal-daily (lactose used as carrier)]. Upon harvest at a federally inspected slaughter facility, epithelium samples were collected from the cranial sac of the rumen, immersed in a 70% alcohol, and preserved under refrigeration (5°C). Ruminal tissue fragments were trimmed (1cm2), in which papillae were completely counted within the fragment, followed by the removal of 12 random individual papillae for further area measurement using electronic scanning (ImageJ). Data were analyzed using the GLIMMIX procedure of SAS and pen was considered the experimental unit with the fixed effect of treatment and the random effect of block. Regardless of treatments, average papillae area in cm2 (P = 0.39; control: 0.38, DFM mixture-A: 0.40, DFM mixture-B: 0.37), papillae number within fragment (P = 0.70; control: 47, DFM mixture-A: 52, DFM mixture-B: 49), absorptive surface area in cm2/fragment cm2 (P = 0.87; control: 37, DFM mixture-A: 39, DFM mixture-B: 38), and ruminal absorptive surface area (P = 0.74; control: 97%, DFM mixture-A: 97%, DFM mixture-B: 97%) were not affected compared with control. The addition of DFM mixtures did not seem to affect ruminal papillae physical aspect, although the long-term effects of dietary inclusion of live bacteria on ruminal morphology needs to be further evaluated.

PSVIII-18 Nutrient Digestibility of Feedlot Beef Yearling Steers Offered Finishing Diets with Direct fed Microbial Mixtures

Abstract The effects of dietary inclusion of live bacteria on feedlot beef cattle apparent total tract nutrient digestibility were evaluated. Crossbred-Angus yearling steers (n = 192; initial BW = 409 kg ± 8 kg) were blocked by body weight (BW) and randomly assigned into 48 pens (4 steers/pen; 16 pens/treatment) following a randomized complete block design. A steam-flaked corn-based fishing diet was offered ad libitum once daily containing the following treatments: 1) Control, in which no direct fed microbial (DFM) was offered (lactose as carrier only); 2 and 3) Probiotic mixtures at distinct concentrations [Mixture A and B, at 2g/animal-daily (lactose used as carrier)]. Orts DM were quantified (if any) daily and subtracted from total dietary DM offered to calculate DM intake. Fecal samples were collected twice daily (0700 and 1700 h) for 5 consecutive days (d 68 to 72) from at least 3 steers per pen, while feed samples were collected daily. Samples were composited within period, dehydrated (55°C), and ground (1mm) for further analyses. Total fecal output was estimated with a dietary internal marker (288-h indigestible NDF) and used to calculate nutrient digestibility. Data were analyzed using the GLIMMIX procedure of SAS and pen was considered the experimental unit. Intakes of DM, OM, NDF, and ADF during the digestibility assessment were not affected (P ≥ 0.13) by treatments. Steers offered DFM mixture-A tended to increase digestibility of DM (P = 0.07; 79.3 vs. 77.1%), NDF (P = 0.10; 56.9 vs. 51.6%), and hemicellulose (P = 0.08; 59.4 vs. 53.9%) compared with control, while digestibility of ADF was not affected (P = 0.24) by treatment. The DFM mixture-A seemed to positively affect apparent digestibility of nutrients in steers consuming a steam-flaked corn-based finishing diet, in which improvements in the fiber fraction digestion seemed to be a meaningful contributor.

Quantitative and qualitative characterization of commercially available oral suspension of probiotic products containing Bacillus Clausii spores

Probiotics contain beneficial live bacteria that confer several health benefits to the host. For the past 50 years, spore-forming Bacillus species have been used in the form of probiotics. Among these, Bacillus clausii strains are used for the management of acute and antibiotic-associated diarrhoea. In the present work, we have evaluated the asserted label information on randomly chosen commercial Bacillus clausii spore suspension of probiotic products. The quality and number of viable bacteria were evaluated based on the colony count, antibiotic resistance, and hemolytic activity assays. The colony fingerprinting and 16S rRNA gene-sequencing techniques were used to confirm the presence of a univariate strain (Bacillus clausii). Our results corroborated the label count of 2 × 109 CFU/5 mL in BACIPRO®, ENTEROGERMINA®, and TUFPRO® products. However, vegetative spore count was not found to match with the given label count in BENEGUT®, PROALANA-B®, β-LOCK®, and PROCILLUS® Bacillus clausii brands. In the hemolytic activity assay, except for β-LOCK®, the other 6 products showed gamma-hemolysis activity. Bacillus clausii isolated from all 7 probiotic products demonstrated resistance to several broad-spectrum antibiotics. The 16S rRNA gene-sequencing data detected genera of Bacillus and Bacillus clausii strain in the BACIPRO®, ENTEROGERMINA®, PROALANA-B®, BENEGUT®, and TUFPRO® products; however, Ralstonia mannitolilytica and Paenibacillus dendritiformis species were identified in β-LOCK® and PROCILLUS®, respectively. As correct label information was observed only in BACIPRO®, ENTEROGERMINA®, and TUFPRO® products, it is proposed that a more stringent quality check would minimize the possibility of mismatch concerning the label information.

Protocol: a simple method for biosensor visualization of bacterial quorum sensing and quorum quenching interaction on Medicago roots

BackgroundDefining interactions of bacteria in the rhizosphere (encompassing the area near and on the plant root) is important to understand how they affect plant health. Some rhizosphere bacteria, including plant growth promoting rhizobacteria (PGPR) engage in the intraspecies communication known as quorum sensing (QS). Many species of Gram-negative bacteria use extracellular autoinducer signal molecules called N-acyl homoserine lactones (AHLs) for QS. Other rhizobacteria species, including PGPRs, can interfere with or disrupt QS through quorum quenching (QQ). Current AHL biosensor assays used for screening and identifying QS and QQ bacteria interactions fail to account for the role of the plant root.MethodsMedicago spp. seedlings germinated on Lullien agar were transferred to soft-agar plates containing the broad-range AHL biosensor Agrobacterium tumefaciens KYC55 and X-gal substrate. Cultures of QS and QQ bacteria as well as pure AHLs and a QQ enzyme were applied to the plant roots and incubated for 3 days.ResultsWe show that this expanded use of an AHL biosensor successfully allowed for visualization of QS/QQ interactions localized at the plant root. KYC55 detected pure AHLs as well as AHLs from live bacteria cultures grown directly on the media. We also showed clear detection of QQ interactions occurring in the presence of the plant root.ConclusionsOur novel tri-trophic system using an AHL biosensor is useful to study QS interspecies interactions in the rhizosphere.

Flavobacterial exudates disrupt cell cycle progression and metabolism of the diatom Thalassiosira pseudonana

Phytoplankton and bacteria form the base of marine ecosystems and their interactions drive global biogeochemical cycles. The effects of bacteria and bacteria-produced compounds on diatoms range from synergistic to pathogenic and can affect the physiology and transcriptional patterns of the interacting diatom. Here, we investigate physiological and transcriptional changes in the marine diatom Thalassiosira pseudonana induced by extracellular metabolites of a known antagonistic bacterium Croceibacter atlanticus. Mono-cultures of C. atlanticus released compounds that inhibited diatom cell division and elicited a distinctive morphology of enlarged cells with increased chloroplast content and enlarged nuclei, similar to what was previously observed when the diatom was co-cultured with live bacteria. The extracellular C. atlanticus metabolites induced transcriptional changes in diatom pathways that include recognition and signaling pathways, cell cycle regulation, carbohydrate and amino acid production, as well as cell wall stability. Phenotypic analysis showed a disruption in the diatom cell cycle progression and an increase in both intra- and extracellular carbohydrates in diatom cultures after bacterial exudate treatment. The transcriptional changes and corresponding phenotypes suggest that extracellular bacterial metabolites, produced independently of direct bacterial-diatom interaction, may modulate diatom metabolism in ways that support bacterial growth.

The Efficacy of Colistin Combined with Amikacin or Levofloxacin against Pseudomonas aeruginosa Biofilm Infection.

ABSTRACTPseudomonas aeruginosa (PA) biofilm infection is clinically prevalent and difficult to eradicate. In the present work, we aimed to evaluate the in vitro and in vivo efficacy of colistin (COL)-based combinations against PA biofilm. MICs and fractional inhibitory concentration indexes (FICIs) of four antibiotics (COL, amikacin, levofloxacin, and meropenem) to bioluminescent strain PAO1, carbapenem-resistant PAO1 (CRPAO1), and clinically isolated strains were assessed. Minimal biofilm eradication concentrations (MBECs) of monotherapy and combinations were examined by counting the live bacteria in biofilm, accompanied by visual confirmation using confocal laser-scanning microscopy. An animal biofilm infection model was established by implanting biofilm subcutaneously, and the therapeutic effect was evaluated according to the change in luminescence through a live animal bio-photonic imaging system. In vitro, even combined with 4 or 8 mg/L COL, meropenem needed to reach 128 or 256 mg/L to eradicate the biofilm. Moreover, 2 mg/L COL combined with 32 mg/L amikacin or 4-8 mg/L levofloxacin could kill the PAO1 and CRPAO1 in biofilm within 24 h. In vivo, COL combined with amikacin or levofloxacin could shorten the eradication time of biofilm than monotherapy. For PAO1 biofilm, combination therapy could eradicate the biofilm in all mice on the 5th day, whereas monotherapy only eradicated biofilms in almost half of the mice. For CRPAO1 biofilm, the biofilm eradication rate on the 6th day in the COL+ amikacin, amikacin, or COL alone regimen was 90%, 10%, or 40%, respectively. COL combined with levofloxacin did not show a better effect than each individual antibiotic. COL-based combinations containing levofloxacin or amikacin were promising choices for treating PA biofilm infection.IMPORTANCE Infections associated with PA biofilm formation are extremely challenging. When monotherapy fails to achieve optimal efficacy, combination therapy becomes the last option. After evaluating multiple drug combinations through a series of experiments in vitro and in vivo, we confirmed that colistin-based combinations containing levofloxacin or amikacin were promising choices for treating PA biofilm infection. The efficacy of these combinations derives from the different bactericidal mechanisms and the bacterial susceptibility to each antibiotic. This study provided a new regimen to solve the incurable problem of biofilm by using COL combined with other antibiotics.

Chronic suppurative otitis media causes macrophage-associated sensorineural hearing loss

BackgroundChronic suppurative otitis media (CSOM) is the most common cause of permanent hearing loss in children in the developing world. A large component of the permanent hearing loss is sensory in nature and our understanding of the mechanism of this has so far been limited to post-mortem human specimens or acute infection models that are not representative of human CSOM. In this report, we assess cochlear injury in a validated Pseudomonas aeruginosa (PA) CSOM mouse model.MethodsWe generated persisters (PCs) and inoculated them into the mouse middle ear cavity. We tracked infection with IVIS and detected PA using RT-PCR. We assessed cochlear damage and innate immunity by Immunohistochemistry. Finally, we evaluated cytokines with multiplex assay and quantitative real-time PCR.ResultsWe observed outer hair cell (OHC) loss predominantly in the basal turn of the cochlear at 14 days after bacterial inoculation. Macrophages, not neutrophils are the major immune cells in the cochlea in CSOM displaying increased numbers and a distribution correlated with the observed cochlear injury. The progression of the morphological changes suggests a transition from monocytes into tissue macrophages following infection. We also show that PA do not enter the cochlea and live bacteria are required for cochlear injury. We characterized cytokine activity in the CSOM cochlea.ConclusionsTaken together, this data shows a critical role for macrophages in CSOM-mediated sensorineural hearing loss (SNHL).