Culturing and Molecular Approaches for Identifying Microbiota Taxa Impacting Children's Obesogenic Phenotypes Related to Xenobiotic Dietary Exposure.

Integrated data from molecular and improved culturomics studies might offer holistic insights on gut microbiome dysbiosis triggered by xenobiotics, such as obesity and metabolic disorders. Bisphenol A (BPA), a dietary xenobiotic obesogen, was chosen for a directed culturing approach using microbiota specimens from 46 children with obesity and normal-weight profiles. In parallel, a complementary molecular analysis was carried out to estimate the BPA metabolising capacities. Firstly, catalogues of 237 BPA directed-cultured microorganisms were isolated using five selected media and several BPA treatments and conditions. Taxa from Firmicutes, Proteobacteria, and Actinobacteria were the most abundant in normal-weight and overweight/obese children, with species belonging to the genera Enterococcus, Escherichia, Staphylococcus, Bacillus, and Clostridium. Secondly, the representative isolated taxa from normal-weight vs. overweight/obese were grouped as BPA biodegrader, tolerant, or resistant bacteria, according to the presence of genes encoding BPA enzymes in their whole genome sequences. Remarkably, the presence of sporobiota and concretely Bacillus spp. showed the higher BPA biodegradation potential in overweight/obese group compared to normal-weight, which could drive a relevant role in obesity and metabolic dysbiosis triggered by these xenobiotics.

Identification of mcr-1 and a novel chloramphenicol resistance gene catT on an integrative and conjugative element in an Actinobacillus strain of swine origin.

The aim of this study was to characterize a mcr-1-carrying integrative and conjugative element (ICE) in a novel Pasteurellaceae-like bacteria of swine origin. The mcr-1-positive GY-402 strain, recovered from a pig fecal sample, was subjected to whole genome sequencing with the combination of Illumina Hiseq and MinION platforms. Genome-based taxonomy revealed that strain GY-402 exhibited highest ANI value (84.89 %) to Actinobacillus succinogenes, which suggested that it represented a novel Actinobacillus species. Sequence analysis revealed that mcr-1 was clustered with eight other resistance genes in the MDR region of a novel ICE element, named ICEAsp1. Inverse PCR and mating assays showed that ICEAsp1 is active and transferrable. In addition, six circular forms mediated by four ISApl1 elements were detected with different inverse PCR sets, indicating that flexible composite transposons could be formed by pairwise combinations of multiple IS copies. Cloning experiment and phylogenetic analysis revealed that the novel Cat protein, designated CatT, belongs to type-A family and confers resistance to chloramphenicol. In conclusion, this is, to the best of our knowledge, the first report of mcr-1 gene on ICE structure and also in Pasteurellaceae bacteria. The diverse composite transposons mediated by multicopy IS elements may facilitate the dissemination of different resistance genes.

From Antibacterial to Antitumour Agents: A Brief Review on The Chemical and Medicinal Aspects of Sulfonamides.

Sulfonamides have been in clinical use for many years, and the development of bioactive substances containing the sulfonamide subunit has grown steadily in view of their important biological properties such as antibacterial, antifungal, antiparasitic, antioxidant, and antitumour properties. This review addresses the medicinal chemistry aspects of sulfonamides; covering their discovery, the structure- activity relationship and the mechanism of action of the antibacterial sulfonamide class, as well as the physico-chemical and pharmacological properties associated with this class. It also provides an overview of the various biological activities inherent to sulfonamides, reporting research that emphasises the importance of this group in the planning and development of bioactive substances, with a special focus on potential antitumour properties. The synthesis of sulfonamides is considered to be simple and provides a diversity of derivatives from a wide variety of amines and sulfonyl chlorides. The sulfonamide group is a non-classical bioisostere of carboxyl groups, phenolic hydroxyl groups and amide groups. This review highlights that most of the bioactive substances have the sulfonamide group, or a related group such as sulfonylurea, in an orientation towards other functional groups. This structural characteristic was observed in molecules with distinct antibacterial activities, demonstrating a clear structure-activity relationship of sulfonamides. This short review sought to contextualise the discovery of classic antibacterial sulfonamides and their physico-chemical and pharmacological properties. The importance of the sulfonamide subunit in Medicinal Chemistry has been highlighted and emphasised, in order to promote its inclusion in the planning and synthesis of future drugs.

Continuous Succinic Acid Fermentation by Actinobacillus Succinogenes: Assessment of Growth and Succinic Acid Production Kinetics.

Succinic acid is one of the most interesting platform chemicals that can be produced in a biorefinery approach. The paper reports the characterization of the growth kinetics of Actinobacillus succinogenes DSM 22257 using glucose as carbon source. Tests were carried out in a continuous bioreactor operated under controlled pH. Under steady-state conditions, the conversion process was characterized in terms of concentration of glucose, cells, acids, and pH. The effects of acid-succinic, acetic, and formic-concentration in the medium on fermentation performance were investigated. The fermentation was interpreted according to several models characterized by substrate and product inhibition. The selected kinetic model of biomass growth and of metabolite production described the microorganism growth rate under a broad interval of operating conditions. Under the investigated operating conditions, results pointed out that: no substrate inhibition was observed; acetic acid did not inhibit the cell growth and succinic acid production.

Multifocal suppurative granuloma caused by Actinobacillus lignieresii in the peritoneum of a beef steer.

An imported crossbred Angus beef steer aged eight to twelve months died suddenly on the eighth day of a quarantine period in Japan. Gross examination showed the peritoneum and mesentery consisted of numerous nodules of various sizes. Histological examination revealed chronic suppurative granulomatous peritonitis with eosinophilic rosettes surrounding colonies of Gram-negative bacilli. The bacteria isolated from the nodules were confirmed to be Actinobacillus lignieresii based on the results of 16S rRNA gene sequencing and immunohistochemistry. Antibiotic sensitivity testing showed that the isolate was resistant to penicillin. Thus, a diagnosis of atypical actinobacillosis caused by A. lignieresii was made.

Succinic acid production from lignocellulosic hydrolysate by Basfia succiniciproducens.

The production of chemicals alongside fuels will be essential to enhance the feasibility of lignocellulosic biorefineries. Succinic acid (SA), a naturally occurring C4-diacid, is a primary intermediate of the tricarboxylic acid cycle and a promising building block chemical that has received significant industrial attention. Basfia succiniciproducens is a relatively unexplored SA-producing bacterium with advantageous features such as broad substrate utilization, genetic tractability, and facultative anaerobic metabolism. Here B. succiniciproducens is evaluated in high xylose-content hydrolysates from corn stover and different synthetic media in batch fermentation. SA titers in hydrolysate at an initial sugar concentration of 60g/L reached up to 30g/L, with metabolic yields of 0.69g/g, and an overall productivity of 0.43g/L/h. These results demonstrate that B. succiniciproducens may be an attractive platform organism for bio-SA production from biomass hydrolysates.

Enhanced succinic acid production from corncob hydrolysate by microbial electrolysis cells.

In this study, Actinobacillus succinogenes NJ113 microbial electrolysis cells (MECs) were used to enhance the reducing power responsible for succinic acid production from corncob hydrolysate. During corncob hydrolysate fermentation, electric MECs resulted in a 1.31-fold increase in succinic acid production and a 1.33-fold increase in the reducing power compared with those in non-electric MECs. When the hydrolysate was detoxified by combining Ca(OH)2, NaOH, and activated carbon, succinic acid production increased from 3.47 to 6.95 g/l. Using a constant potential of -1.8 V further increased succinic acid production to 7.18 g/l. A total of 18.09 g/l of succinic acid and a yield of 0.60 g/g total sugar were obtained after a 60-h fermentation when NaOH was used as a pH regulator. The improved succinic acid yield from corncob hydrolysate fermentation using A. succinogenes NJ113 in electric MECs demonstrates the great potential of using biomass as a feedstock to cost-effectively produce succinate.

Succinic acid production with Actinobacillus succinogenes: rate and yield analysis of chemostat and biofilm cultures.

BACKGROUND: Succinic acid is well established as bio-based platform chemical with production quantities expecting to increase exponentially within the next decade. Actinobacillus succinogenes is by far the most studied wild organism for producing succinic acid and is known for high yield and titre during production on various sugars in batch culture. At low shear conditions continuous fermentation with A. succinogenes results in biofilm formation. In this study, a novel shear controlled fermenter was developed that enabled: 1) chemostat operation where self-immobilisation was opposed by high shear rates and, 2) in-situ removal of biofilm by increasing shear rates and subsequent analysis thereof. RESULTS: The volumetric productivity of the biofilm fermentations were an order of magnitude more than the chemostat runs. In addition the biofilm runs obtained substantially higher yields. Succinic acid to acetic acid ratios for chemostat runs were 1.28±0.2 g.g(-1), while the ratios for biofilm runs started at 2.4 g.g(-1) and increased up to 3.3 g.g(-1) as glucose consumption increased. This corresponded to an overall yield on glucose of 0.48±0.05 g.g(-1) for chemostat runs, while the yields varied between 0.63 g.g(-1) and 0.74 g.g(-1) for biofilm runs. Specific growth rates (µ) were shown to be severely inhibited by the formation of organic acids, with µ only 12% of µ(max) at a succinic acid titre of 7 g.L(-1). Maintenance production of succinic acid was shown to be dominant for the biofilm runs with cell based production rates (extracellular polymeric substance removed) decreasing as SA titre increases. CONCLUSIONS: The novel fermenter allowed for an in-depth bioreaction analysis of A. succinogenes. Biofilm cells achieve higher SA yields than suspended cells and allow for operation at higher succinic acid titre. Both growth and maintenance rates were shown to drastically decrease with succinic acid titre. The A. succinogenes biofilm process has vast potential, where self-induced high cell densities result in higher succinic acid productivity and yield.

Immobilization of Actinobacillus succinogenes by adhesion or entrapment for the production of succinic acid.

The production of succinic acid was studied with entrapped and adsorbed Actinobacillus succinogenes. The adsorption of fermentation products (organic acids in the concentration range of 1-20 g/L) on different supports was evaluated. It was found that succinic acid was adsorbed in small quantities on diatomite and zeolite (12.6 mg/g support). The highest production of succinic acid was achieved with A. succinogenes entrapped in agar beads. Batch fermentations with immobilized cells were carried out with glucose concentrations ranging from 20 to 80 g/L. Succinic acid (43.4 g/L) was obtained from 78.3g/L glucose, and a high productivity (2.83 g/Lh) was obtained with a glucose concentration of 37.6g/L. For repeated batch fermentations (5 cycles in 72 h) with immobilized cells in agar, the total glucose consumed was 147.55 g/L, while the production of succinic acid was 107 g/L. Immobilized cells reduced significantly the fermentation time, yield, productivity and final concentration of succinic acid.

Antibacterial effects and biocompatibility of titanium surfaces with graded silver incorporation in titania nanotubes.

Most commercial dental implants are made of titanium (Ti) because Ti possesses excellent properties such as osseointegration. However, many types of Ti products still suffer from insufficient antibacterial capability and bacterial infection after surgery remains one of the most common and intractable complications. In this study, a dual process encompassing anodization and silver plasma immersion ion implantation (Ag PIII) is utilized to produce titania nanotubes (TiO2-NTs) containing Ag at different sites and depths. The concentration and depth of the incorporated Ag can be tailored readily by changing the PIII parameters. The Ag-embedded TiO2-NTs which retain the nanotubular morphology are capable of sterilizing oral pathogens as opposed to pure Ti plates and pristine TiO2-NTs. Biological assays indicate that the in vitro and in vivo biocompatibility of the sample plasma-implanted at a lower voltage of 0.5 kV (NT-Ag-0.5) is significantly compromised due to the large amount of surface Ag. On the other hand, the sample implanted at 1 kV (NT-Ag-1.0) exhibits unimpaired effects due to the smaller surface Ag accumulation. Sample NT-Ag-1.0 is further demonstrated to possess sustained antibacterial properties due to the large embedded depth of Ag and the technique and resulting materials have large potential in dental implants.

Succinic acid production from sucrose by Actinobacillus succinogenes NJ113.

In this study, sucrose, a reproducible disaccharide extracted from plants, was used as the carbon source for the production of succinic acid by Actinobacillus succinogenes NJ113. During serum bottle fermentation, the succinic acid concentration reached 57.1g/L with a yield of 71.5%. Further analysis of the sucrose utilization pathways revealed that sucrose was transported and utilized via a sucrose phosphotransferase system, sucrose-6-phosphate hydrolase, and a fructose PTS. Compared to glucose utilization in single pathway, more pathways of A. succinogenes NJ113 are dependent on sucrose utilization. By changing the control strategy in a fed-batch culture to alleviate sucrose inhibition, 60.5g/L of succinic acid was accumulated with a yield of 82.9%, and the productivity increased by 35.2%, reaching 2.16g/L/h. Thus utilization of sucrose has considerable potential economics and environmental meaning.

Succinic acid production by Actinobacillus succinogenes NJ113 using corn steep liquor powder as nitrogen source.

In this study, corn steep liquor powder (CSL) was used as nitrogen source to replace the relatively costly yeast extract typically used for the production of succinic acid with Actinobacillus succinogenes NJ113. Moreover, when heme was added to the fermentation medium and the culture was agitated at a low speed, a maximum succinic acid concentration of 37.9 g/l was obtained from a glucose concentration of 50 g/l, and a productivity of 0.75 g/l/h was achieved. These yields are almost as high as for fermentation with glucose and yeast extract. These results suggest that heme-supplemented CSL may be a suitable alternative nitrogen source for a cost-effective method of producing succinic acid with A. succinogenes NJ113 while consuming less energy than previous methods.

Succinic acid production from cellobiose by Actinobacillus succinogenes.

In this study, cellobiose, a reducing disaccharide was used to produce succinic acid by Actinobacillus succinogenes NJ113. A final succinic acid concentration of 30.3g/l with a yield of 67.8% was achieved from an initial cellobiose concentration of 50 g/l via batch fermentation in anaerobic bottles. The cellobiose uptake mechanism was investigated and the results of enzyme assays revealed that the phosphoenolpyruvate phosphotransferase system (PEP-PTS) played an important role in the cellobiose uptake process. In batch fermentation with 18 g/l of cellobiose and 17 g/l of other sugars from sugarcane bagasse cellulose hydrolysates, a succinic acid concentration of 20.0 g/l was obtained, with a corresponding yield of 64.7%. This study found that cellobiose from incomplete hydrolysis of cellulose could be a potential carbon source for economical and efficient succinic acid production by A. succinogenes.

Synergistic antibacterial effect between silibinin and antibiotics in oral bacteria.

Silibinin is a composition of the silymarin group as a hepatoprotective agent, and it exhibits various biological activities, including antibacterial activity. In this study, the antibacterial activities of silibinin were investigated in combination with two antimicrobial agents against oral bacteria. Silibinin was determined with MIC and MBC values ranging from 0.1 to 3.2 and 0.2 to 6.4 µg/mL, ampicillin from 0.125 to 64 and 0.5 to 64 µg/mL, gentamicin from 2 to 256 and 4 to 512 µg/mL, respectively. The ranges of MIC(50) and MIC(90) were 0.025-0.8 µg/mL and 0.1-3.2 µg/mL, respectively. The antibacterial activities of silibinin against oral bacteria were assessed using the checkerboard and time-kill methods to evaluate the synergistic effects of treatment with ampicillin or gentamicin. The results were evaluated showing that the combination effects of silibinin with antibiotics were synergistic (FIC index <0.5) against all tested oral bacteria. Furthermore, a time-kill study showed that the growth of the tested bacteria was completely attenuated after 2-6 h of treatment with the MBC of silibinin, regardless of whether it was administered alone or with ampicillin or gentamicin. These results suggest that silibinin combined with other antibiotics may be microbiologically beneficial and not antagonistic.

Succinic acid production by Actinobacillus succinogenes using hydrolysates of spent yeast cells and corn fiber.

The enzymatic hydrolysate of spent yeast cells was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113, using corn fiber hydrolysate as a carbon source. When spent yeast cell hydrolysate was used directly as a nitrogen source, a maximum succinic acid concentration of 35.5 g/l was obtained from a glucose concentration of 50 g/l, with a glucose utilization of 95.2%. Supplementation with individual vitamins showed that biotin was the most likely factor to be limiting for succinic acid production with spent yeast cell hydrolysate. After supplementing spent yeast cell hydrolysate and 90 g/l of glucose with 150 µg/l of biotin, cell growth increased 32.5%, glucose utilization increased 37.6%, and succinic acid concentration was enhanced 49.0%. As a result, when biotin-supplemented spent yeast cell hydrolysate was used with corn fiber hydrolysate, a succinic acid yield of 67.7% was obtained from 70.3 g/l of total sugar concentration, with a productivity of 0.63 g/(l h). Our results suggest that biotin-supplemented spent yeast cell hydrolysate may be an alternative nitrogen source for the efficient production of succinic acid by A. succinogenes NJ113, using renewable resources.

Kinetic evaluation of products inhibition to succinic acid producers Escherichia coli NZN111, AFP111, BL21, and Actinobacillus succinogenes 130Z T.

Succinic acid is one of the platform compounds and its production via natural feedstocks has drawn worldwide concerns. To evaluate the inhibitory effects of fermentation products on the growth of Actinobacillus succinogenes 130Z(T) and Escherichia coli NZN111, AFP111, BL21, fermentations with addition of individual products in medium were carried out. The cell growth was inhibited when the concentrations of formate, acetate, lactate, and succinate were at range of 8.8-17.6 g/L, 10-40 g/L, 9-18 g/L, and 10-80 g/L, respectively. For these two species of bacteria, E. coli was more resistant to acid products than A. succinogenes, while both endured succinate rather than by-products. As a result of end product inhibition, succinate production yield by A. succinogenes decreased from 1.11 to 0.49 g/g glucose. Logistic and Monod mathematical models were presented to simulate the inhibition kinetics. The Logistic model was found more suitable for describing the overall synergistic inhibitory effects.

Efficient conversion of crop stalk wastes into succinic acid production by Actinobacillus succinogenes.

Succinic acid is valued as a key platform chemical for use in a variety of synthetic applications. Efficient biosynthesis of succinic acid from renewable biomass resource is reported in this paper. Batch fermentations were carried out to analyze influence of several carbon sources on succinic acid production from feedstock wastes by Actinobacillus succinogenes BE-1. Crop stalk wastes, including corn stalk and cotton stalk, were enzymatically converted into a carbohydrate-rich feedstock, obtaining glucose concentrations approaching 65-80% of the total reducing sugar. For the anaerobic batch cultivation with cotton stalk hydrolysates, the production of succinic acid was 15.8 g l(-1) with a high yield of 1.23 g per g glucose. Glucose and xylose were utilized at same time, while cellubiose was not consumed until glucose and xylose were completely consumed.

Uptake pathways of anionic and cationic photosensitizers into bacteria.

The effect of divalent cations (calcium and magnesium) and a permeabilizing agent (EDTA) on the uptake of a cationic photosensitizer (PS), methylene blue (MB), and two anionic PSs, rose bengal (RB) and indocyanine green (ICG), by Gram-positive Enterococcus faecalis and Gram-negative Actinobacillus actinomycetemcomitans was examined. The possible roles of multidrug efflux pumps and protein transporters in photosensitizer uptake were assessed in E. faecalis cells by studies using an efflux pump inhibitor (verapamil) and trypsin treatment respectively. Divalent cations enhanced the uptake and photodynamic inactivation potential of both RB and ICG in E. faecalis and A. actinomycetemcomitans, while they decreased the uptake and bacterial killing by MB. Verapamil increased the uptake of RB (possibly due to efflux pump inhibition), whereas trypsin treatment resulted in significant decrease in RB and ICG uptake. The results suggested that the uptake of anionic PSs by bacterial cells may be mediated through a combination of electrostatic charge interaction and by protein transporters, while the uptake of cationic PSs, as previously reported, is mediated by electrostatic interactions and self promoted uptake pathways.

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes.

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l(-1). In batch fermentation, 45.5 g l(-1) succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l(-1) of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l(-1) at a rate of 1.21 g l(-1) h(-1) after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.

Antimicrobial resistance profile of Actinobacillus pleuropneumoniae and Actinobacillus porcitonsillarum.

A total of 83 Actinobacillus pleuropneumoniae and 58 Actinobacillus porcitonsillarum strains collected from slaughtered pigs in Switzerland were screened for susceptibility to 20 antimicrobial agents by MIC determinations. Resistance to sulfamethoxazole, the combination sulfamethoxazole-trimethoprim, tiamulin, tilmicosin, tetracycline, penicillin and ampicillin were found. A few A. porcitonsillarum isolates displayed decreased susceptibility to enrofloxacin. PCR analysis revealed the presence of the sul2 gene in approximately one-fifth of the sulfonamide-resistant A. pleuropneumoniae and A. porcitonsillarum isolates. The tetracycline-resistant A. pleuropneumoniae harbored tet(B) and tet(H), whereas the tetracycline-resistant A. porcitonsillarum isolates harbored the tet(B) gene. The penicillin and ampicillin-resistant A. pleuropneumoniae and A. porcitonsillarum harbored the bla(ROB-1) gene.