Bacillus spizizenii DN and microbial consortia biostimulation followed by gamma irradiation for efficient textile wastewater treatment.

Textile wastewater still poses a huge environmental problem due to its high water consumption and high effluent release that is full of toxic chemicals. In the present study, different approaches were studied to layout an operating procedure for textile wastewater treatment in order to obtain treated wastewater that is safe for non-potable uses. Our approach depended on (1) co-substrate to biostimulate indigenous microbial textile wastewater community by adding Tryptone Soy Broth (TSB) and TSB supplemented with 1% glucose, (2) co-culture (bioaugmentation) with Bacillus spizizenii DN cells (previously isolated, identified and characterized as efficient decolorizing bacteria), and (3) co-metabolites using Bacillus spizizenii DN metabolites. The obtained results show that using Bacillus spizizenii DN cells resulted in 97.78% decolorization while adding Bacillus spizizenii DN metabolites resulted in 82.92% decolorization, both after 48 h incubation under microaerophilic conditions. The phyla identified for all treatments were Bacteroidota, Firmicutes, and Proteobacteria. The dynamic changes in the bacteria showed that both Clostridium and Acinetobacter disappeared for co-substrate, co-culture, and co-metabolite cultures. While Alkalibacterium and Stenotrophomonas appeared after adding Bacillus spizizenii DN cells, Flavobacterium increased for co-substrate and co-metabolic cultures while iron reducing bacteria appeared only for co-metabolic cultures. The use of 25 kGy gamma irradiation as a sterilization dose post bioremediation ensured safe use of treated wastewater. This was confirmed by cytotoxicity assay; the obtained IC50 tested on BJ fibroblasts obtained from skin showed that gamma irradiated treated wastewater are about 80.1% less toxic than non-irradiated treated wastewater. We conclude that (1) we can use combined bioaugmentation and biostimulation as initial steps for in situ bioremediation in collection tanks and that (2) the proposed protocol for bioremediation of industrial wastewater should be tailored based on the required application and level of safety needed for re-use.

Inhibition of acetic acid-induced colitis in rats by new Pediococcus acidilactici strains, vitamin producers recovered from human gut microbiota.

Our aim was to isolate, identify and characterize probiotic bacteria as vitamin producers in particular B2 and B9. 150 human fecal samples were collected and used for isolation of vitamin producers-probiotics. 49 isolates were chosen for screening their genome by PCR for the presence of riboflavin and folic acid genes. As a result, three isolates were selected and their production of the B2 and B9 were confirmed by HPLC. The three isolates were identified on species level by sequencing their 16S rRNA gene which showed 100% identical to strains of Pediococcus acidilactici. Thus, they were named as P. acidilactici WNYM01, P. acidilactici WNYM02, P. acidilactici WNYM03 and submitted to the Genbank database with accession numbers. They met the probiotic criteria by expressing 90-95% survival rate at pH (2.0-9.0) and bile salt up to 2% for 3 h in addition to their antimicrobial activity against gram positive and negative microorganisms. They also showed no hemolytic activity and common pattern for antibiotic susceptibility. Our three strains were tested individually or in mixture in vivo on rat colitis model compared to ulcerative group. The strains were administrated orally to rats in daily dose containing CFU 109 for 14 days then followed by induction of colitis using acetic acid then the oral administration was continued for more four days. The histology results, the anti-inflammatory and anti-oxidative stress biomarkers showed the protective role of the strains compared to the ulcerative group. As a conclusion, we introduce novel three probiotic candidates for pharmaceutical preparations and health applications.

Complete genome sequence and comparative analysis of two potential probiotics Bacillus subtilis isolated from honey and honeybee microbiomes.

BACKGROUND: We have previously isolated Bacillus subtilis HMNig-2 and MENO2 strains, from honey and the honeybee gut microbiome respectively, and demonstrated these strains to produce levansucrase with potential probiotics characteristics. Here we report their complete genome sequence and comparative analysis with other and other B. subtilis strains. RESULTS: The complete genome sequences of Bacillus subtilis HMNig-2 and MENO2 were de novo assembled from MiSeq paired-end sequence reads and annotated using the RAST tool. Whole-genome alignments were performed to identify functional differences associated with their potential use as probiotics. CONCLUSIONS: The comparative analysis and the availability of the genome sequence of these two strains will provide comprehensive analysis about the diversity of these valuable Bacillus strains and the possible impact of the environment on bacterial evolution. SIGNIFICANCE AND IMPACT OF STUDY: We introduce complete genome sequence of two new B. subtilis strains HMNig-2 and MENO2 with probiotic potential and as cell factories for the production of levan and other valuable components for pharmaceutical and industrial applications.

Inhibition of Clostridium difficile in Mice Using a Mixture of Potential Probiotic Strains Enterococcus faecalis NM815, E. faecalis NM915, and E. faecium NM1015: Novel Candidates to Control C. difficile Infection (CDI).

This study is aimed at the isolation, identification, and characterization of potential probiotic strains capable of inhibiting Clostridium difficile in vitro and in vivo. Twenty isolates were isolated from infant fecal samples and screened against C. difficile using their cell-free supernatant. Only three isolates showed maximum inhibition from 56.05 to 60.60%, thus they were characterized for probiotic properties and safety. The results obtained approved their tolerance to the gastrointestinal tract conditions and safety profile. They were identified by sequencing 16S rRNA as Enterococcus faecalis NM815, E. faecalis NM915, and Enterococcus faecium NM1015. For in vivo evaluation, a viable mixture of these three strains (109 CFU/mL) was administrated to a group of mice (treated group) in daily dose for 14 days, then followed by challenge with viable C. difficile (105 CFU/mL) in daily dose for 7 days, then a second administration of a viable mixture of the three strains was done daily for 7 days. In addition, the control group was administered PBS buffer only and the untreated group received PBS buffer instead of the probiotic mixture before and after the challenge with C. difficile. The results obtained from histological analysis confirmed the effectiveness of our three potential probiotic strains which expressed inhibition of C. difficile and maintained the structural integrity of the liver and intestinal cells.

Possible correlation between levansucrase production and probiotic activity of Bacillus sp. isolated from honey and honey bee.

Five bacterial isolates from honey and bee gut were selected based on their high levansucrase activity and levan yield which were strongly positively correlated. All isolates showed good tolerance to temperature up to 70 °C, to NaCl up to 3 M and to 0.1% H2O2. They maintained over 59 and 64% survival at pH 9.0 and 2.0 respectively, but showed varying tolerance to 0.1% bile salts and pancreatic enzymes. Most isolates were susceptible to widely used antibiotics, but demonstrated diverse antimicrobial activity. Non hemolytic isolates were identified on the basis of 16S rRNA sequencing as Bacillus subtilis HMNig-2 and B. subtilis MENO2 with 97% homology. They exhibited promising probiotic characteristics and achieved highest levansucrase activity of 94.1 and 81.5 U/mL respectively. Both exhibited highest biofilm formation ability in static microtiter plate assay. Also, they achieved 34 and 26% adhesion respectively to Caco-2cells and had highest free radical scavenging activity of 30.8 and 26.2% respectively. The levans of the two isolates showed good antimicrobial activity against some pathogens and exhibited positive prebiotic effect (prebiotic index >1) with Lactobacillus casei and Lactobacillus reuteri. Results suggest a correlation between levansucrase production, levan yield and pre-probiotic activities of the studied strains.

Serum Interleukin-18 and Its Gene Haplotypes Profile as Predictors in Patients with Diabetic Nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is known as an acute microvascular complexity as a subsequence progression in diabetes mellitus type 1 and 2. Many evidence pointed that the proinflammatory cytokine Interleukin (IL)-18 might be involved in the pathogenesis of DN. AIM: The current study aimed to evaluate the association of serum IL-18 and its promoter gene polymorphisms with diabetic nephropathy. METHODS: This study included 62 diabetic nephropathy patients (DN group) compared to 52 diabetes mellitus patients (DM group). The two groups were subjected to anthropometry assessment, molecular studies including SNP genotyping by RFLP and finally statistical analysis. RESULTS: The assessment of the serum IL-18 level and the frequencies of its allele and haplotype: -137G/C, -607C/A and -656G/T among the DN and DM subjects revealed that -137G allele has significant variation between DN and DM subjects (about 80.8%, P = 0.05) but, no significant variation in -607 or -656 alleles IL-18 gene promoter. CONCLUSION: These data confirm the impact of high serum IL-18 and the haplotype of the polymorphism located in the promoter region of the IL-18 gene with the DN.

Oral immunization of mice with engineered Lactobacillus gasseri NM713 strain expressing Streptococcus pyogenes M6 antigen.

The aim of this in vivo study was to evaluate the effects of a recombinant probiotic strain, Lactobacillus gasseri NM713, which expresses the conserved region of streptococcal M6 protein (CRR6), as an oral vaccine against Streptococcus pyogenes. A dose of 10(9) cells of the recombinant strain in 150 µL PBS buffer was administered orally to a group of mice. One control group received an equivalent dose of Lb. gasseri NM613 (containing the empty plasmid without insert) or and another control group received PBS buffer. Each group contained 30 mice. The immunization protocol was followed on three consecutive days, after which two booster doses were administered at two week intervals. Fecal and serum samples were collected from the mice on Days 18, 32, 46, 58 after the first immunization and Day 0 prior to immunization. Anti-CRR6 IgA and IgG concentrations were measured by ELISA in fecal and sera samples, respectively, to assess immune responses. Vaccination with the recombinant Lb. gasseri NM713 strain induced significant protection after nasal challenge with S. pyogenes, only a small percentage of this group developing streptococcal infection (10%) or dying of it (3.3%) compared with the NM613 and PBS control groups, high percentages of which developed streptococcal infection (43.3% and 46.7%, respectively) and died of it (46.7% and 53%, respectively). These results indicate that recombinant Lb. gasseri NM713 has potential as an oral delivery vaccine against streptococcus group A.

A new lactobacilli in vivo expression system for the production and delivery of heterologous proteins at mucosal surfaces.

Food-grade lactic acid bacteria, such as lactobacilli, represent good candidates for the development of mucosal vectors. Indeed, they are generally recognized as safe microorganisms and some strains display beneficial effects (probiotics). In this study, we described a new lactobacilli in vivo expression (LIVE) system for the production and delivery of therapeutic molecules at mucosal surfaces. The versatility and functionality of this system was successfully validated in several lactobacilli species; furthermore, we assessed in vivo LIVE system in two different mouse models of human pathologies: (i) a model of therapy against intestinal inflammation (inflammatory bowel diseases) and (ii) a model of vaccination against dental caries. We demonstrated that Lactobacillus gasseri expressing the anti-inflammatory cytokine IL-10 under LIVE system efficiently delivered the recombinant protein at mucosal surfaces and display anti-inflammatory effects. In the vaccination model against caries, LIVE system allowed the heterologous expression of Streptococcus mutans antigen GbpB by L. gasseri, leading to a stimulation of the host immune response.

Isolation of Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512 as novel probiotics with immunomodulatory properties.

Probiotics, defined as living bacteria that are beneficial for human health, mainly function through their immunomodulatory abilities. Hence, these microorganisms have proven successful for treating diseases resulting from immune deregulation. The aim of this study was to find novel candidates to improve on and complement current probiotic treatment strategies. Of 60 lactic acid bacterial strains that were isolated from fecal samples of healthy, full-term, breast-fed infants, three were chosen because of their ability to activate human immune cells. These candidates were then tested with regard to immunomodulatory properties, antimicrobial effects on pathogens, required pharmacological properties and their safety profiles. To identify the immunomodulatory structures of the selected isolates, activation of specific innate immune receptors was studied. The three candidates for probiotic treatment were assigned Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512. Compared with the established allergy-protective strain Lactococcus lactis G121, these isolates induced release of similar amounts of IL-12, a potent inducer of T helper 1 cells. In addition, all three neonatal isolates had antimicrobial activity against pathogens. Analysis of pharmacological suitability showed high tolerance of low pH, bile salts and pancreatic enzymes. In terms of safe application in humans, the isolates were sensitive to three antibiotics (chloramphenicol, tetracycline and erythromycin). In addition, the Enterococcus isolates were free from the four major virulence genes (cylA, agg, efaAfs and ccf). Moreover, the isolates strongly activated Toll-like receptor 2, which suggests lipopeptides as their active immunomodulatory structure. Thus, three novel bacterial strains with great potential as probiotic candidates and promising immunomodulatory properties have here been identified and characterized.

Purification and characterization of a cytotoxic neurotoxin-like protein from Naja haje haje venom that induces mitochondrial apoptosis pathway.

This study reported the purification and characterization of a cytotoxic, neurotoxin-like protein derived from the venom of the Egyptian cobra Naja haje haje, Elapidae family, and explored their mechanistic role in the cell death. The protein purification was performed through ion-exchange chromatography and gel-filtration chromatography and was characterized by SDS-PAGE, amino acid sequencing, and mass spectrum analysis. The antitumor activity of Naja haje venom (NHV) and its fractions (NHVI, NHV-Ia, NHV-Ib, NHV-Ic, NHV-II, NHV-III, and NHV-IV) were tested against different human cancer cell lines. The molecular cascade of cell death was explored through evaluation of apoptosis/necrosis ratio, DNA fragmentation, histone deacetylase (HDAC) activity, mitochondrial transmembrane potential (Δψ(m)), cytochrome c release, total caspases, caspase-3, caspase-9, and cell cycle analysis by flow cytometry. Most of the separated fractions possessed variable cytotoxic effect against different cancer cells. The most potent antitumor fraction was NHV-Ic due to its ability to induce DNA damaging and fragmentation that was associated with a significant induction of apoptosis via mitochondrial pathway and disturbed cell cycle phases as well as an inhibition of HDAC activity. NHV-Ic induced the mitochondrial pathway initially by the impairment of Δψ(m) besides the DNA damage and in response to that the mitochondria-released cytochrome c that may in turn activated total caspases, caspase-3 and caspase-9 in lymphoblastic leukemia 1301 cells. The partial amino acid sequencing of NHV-Ic revealed 100, 95.65, and 91.3% homology with the Long neurotoxin 1 from Naja haje anchietae (Angolan cobra), Naja haje haje (Egyptian cobra), and Boulengerina annulata annulata (banded water cobra), respectively.

The bile/arsenite/riboflavin transporter (BART) superfamily.

Secondary transmembrane transport carriers fall into families and superfamilies allowing prediction of structure and function. Here we describe hundreds of sequenced homologues that belong to six families within a novel superfamily, the bile/arsenite/riboflavin transporter (BART) superfamily, of transport systems and putative signalling proteins. Functional data for members of three of these families are available, and they transport bile salts and other organic anions, the bile acid:Na(+) symporter (BASS) family, inorganic anions such as arsenite and antimonite, the arsenical resistance-3 (Acr3) family, and the riboflavin transporter (RFT) family. The first two of these families, as well as one more family with no functionally characterized members, exhibit a probable 10 transmembrane spanner (TMS) topology that arose from a tandemly duplicated 5 TMS unit. Members of the RFT family have a 5 TMS topology, and are homologous to each of the repeat units in the 10 TMS proteins. The other two families [sensor histidine kinase (SHK) and kinase/phosphatase/synthetase/hydrolase (KPSH)] have a single 5 TMS unit preceded by an N-terminal TMS and followed by a hydrophilic sensor histidine kinase domain (the SHK family) or catalytic domains resembling sensor kinase, phosphatase, cyclic di-GMP synthetase and cyclic di-GMP hydrolase catalytic domains, as well as various noncatalytic domains (the KPSH family). Because functional data are not available for members of the SHK and KPSH families, it is not known if the transporter domains retain transport activity or have evolved exclusive functions in molecular reception and signal transmission. This report presents characteristics of a unique protein superfamily and provides guides for future studies concerning structural, functional and mechanistic properties of its constituent members.

The copper transporter (Ctr) family of Cu+ uptake systems.

Copper (Cu(+)) transporters of the Ctr family are sequence diverse eukaryotic proteins that function by an unknown mechanism of action. We have conducted bioinformatic analyses of sequenced Ctr proteins. Multiple paralogues are found in single organisms, and these may be either closely or distantly related to each other. Protein phylogeny generally correlates with organismal source and protein size with proteins of each cluster being derived from a specific eukaryotic kingdom and exhibiting characteristic domain arrangements. Some homologues exhibit repeats of the basic 3 TMS unit. Regions of conserved hydrophobicity and amphipathicity suggest functional roles, particularly for putative TMSs 2 and 3 which possess a nearly fully conserved M X(3) M motif in putative TMS2. We propose that this motif comprises the transmembrane Cu(+)-binding site in oligomeric channels that take up Cu(+) by a passive, membrane potential-dependent mechanism.

Probiotics and prebiotics in human health.

Probiotic bacteria are found in the intestines of humans and other mammals where they provide health benefits to the host. They do so by (1) providing nutrients and cofactors, (2) successfully competing with pathogens, and (3) stimulating host immune responses by producing specific polysaccharides. These bacteria can also alleviate the symptoms of disease-related metabolic disorders. Prebiotics are substances, usually poorly metabolized polysaccharides and oligosaccharides, that cannot be ingested effectively by the animal. They stimulate the growth of intestinal probiotic bacteria, which can utilize these carbohydrates, thereby promoting health of the organism. Genetic engineering has proven useful for the design of probiotic bacteria that counteract the symptoms of genetic and age-related diseases. Can these bacteria be engineered, through human-promoted accelerative evolution, so that they stimulate their own growth and that of other probiotic bacteria so as to crowd pathogens out of the intestine?