Probiotic Potential Analysis and Safety Evaluation of Enterococcus durans A8-1 Isolated From a Healthy Chinese Infant

To evaluate the probiotic characteristics and safety of Enterococcus durans isolate A8-1 from a fecal sample of a healthy Chinese infant, we determined the tolerance to low pH, survival in bile salts and NaCl, adhesion ability, biofilm formation, antimicrobial activity, toxin gene distribution, hemolysis, gelatinase activity, antibiotic resistance, and virulence to Galleria mellonella and interpreted the characters by genome resequencing. Phenotypically, E. durans A8-1 survived at pH 5.0 in 7.0% NaCl and 3% bile salt under aerobic and anaerobic condition. The bacterium had higher adhesion ability toward mucin, collagen, and Bovine Serum Albumin (BSA) in vitro and showed high hydrophobicity (79.2% in chloroform, 49.2% in xylene), auto-aggregation activity (51.7%), and could co-aggregate (66.2%) with Salmonella typhimurium. It had adhesion capability to intestinal epithelial Caco-2 cells (38.74%) with moderate biofilm production and antimicrobial activity against several Gram-positive pathogenic bacteria. A8-1 can antagonize the adhesion of S. typhimurium ATCC14028 on Caco-2 cells to protect the integrity of the cell membrane by detection of lactate dehydrogenase (LDH) and AKP activities. A8-1 also helps the cell relieve the inflammation induced by lipopolysaccharide by reducing the expression of cytokine IL-8 (P = 0.002) and TNF-α (P > 0.05), and increasing the IL-10 (P < 0.001). For the safety evaluation, A8-1 showed no hemolytic activity, no gelatinase activity, and had only asa1 positive in the seven detected virulence genes in polymerase chain reaction (PCR), whereas it was not predicted in the genome sequence. It was susceptible to benzylpenicillin, ampicillin, ciprofloxacin, levofloxacin, moxifloxacin, tigecycline, nitrofurantoin, linezolid, vancomycin, erythromycin, and quinupristin/dalofopine except clindamycin, which was verified by the predicted lasA, lmrB, lmrC, and lmrD genes contributing to the clindamycin resistance. The virulence test of G. mellonella showed that it had toxicity lower than 10% at 1 × 107 CFU. According to the results of these evaluated attributes, E. durans strain A8-1 could be a promising probiotic candidate for applications.

Resistance and virulence distribution in enterococci isolated from broilers reared in two farming systems

Background The impact of enterococci in human health has been growing for the last decades, mainly due to their resistance to several antimicrobial agents. Human consumption of contaminated meat, especially poultry, has been identified as a possible route of transmission. The aim of the present study was to evaluate and compare the antimicrobial resistance profiles and virulence genes of enterococci isolated from Portuguese conventional and free-range broiler farms. Results Antibiotic susceptibility testing showed high frequencies of resistance to tetracycline in both farming systems. Resistance to erythromycin and gentamicin were detected in about half of the isolates. Resistance to penicillin was the less frequently observed and no vancomycin resistant isolates were identified. The majority of the enterococcal isolates, from either farming systems, were resistant to more than one antibiotic, and no statistical associations were found, except for penicillin resistance which associated with the genetic clusters. No differences were found between farming systems regarding the prevalence of tet(M), erm(B), aac (6′)-Ie-aph (2″)-Ia and pbp5 genes, nevertheless pbp5 prevalence was associated with the different genetic clusters. Hemolytic activity was identified in 26.47% of all isolates and gelatinase activity in 50%. The gelE gene was identified in the majority of the isolates, whereas esp and agg genes were rarely detected. The cylA determinant was not detected in any of the isolates. Conclusions Overall, results suggest that similar resistance patterns and virulence genes can be found in both farming systems, though enterococci in free-range conditions should be less prone to acquire further resistance genes.

Cigarette Smoke Condensate Exposure Induces Receptor for Advanced Glycation End-Products (RAGE)-Dependent Sterile Inflammation in Amniotic Epithelial Cells

Maternal smoking is a risk factor of preterm prelabor rupture of the fetal membranes (pPROM), which is responsible for 30% of preterm births worldwide. Cigarettes induce oxidative stress and inflammation, mechanisms both implicated in fetal membranes (FM) weakening. We hypothesized that the receptor for advanced glycation end-products (RAGE) and its ligands can result in cigarette-dependent inflammation. FM explants and amniotic epithelial cells (AECs) were treated with cigarette smoke condensate (CSC), combined or not with RAGE antagonist peptide (RAP), an inhibitor of RAGE. Cell suffering was evaluated by measuring lactate dehydrogenase (LDH) medium-release. Extracellular HMGB1 (a RAGE ligand) release by amnion and choriodecidua explants were checked by western blot. NF-κB pathway induction was determined by a luciferase gene reporter assay, and inflammation was evaluated by cytokine RT-qPCR and protein quantification. Gelatinase activity was assessed using a specific assay. CSC induced cell suffering and HMGB1 secretion only in the amnion, which is directly associated with a RAGE-dependent response. CSC also affected AECs by inducing inflammation (cytokine release and NFκB activation) and gelatinase activity through RAGE engagement, which was linked to an increase in extracellular matrix degradation. This RAGE dependent CSC-induced inflammation associated with an increase of gelatinase activity could explain a pathological FM weakening directly linked to pPROM.

Enzymatic Activity of Psychrotolerant Antarctic Bacteria

The Antarctic region has significant potential to study the biodiversity of microorganisms and to search for bacterial producers of glycolytic and proteolytic enzymes with new properties. The aim was to study the extracellular glycosidase and proteolytic activity of four bacteria strains isolated from black lichens of the cliffs of Galindez Island in the Antarctic. Methods. Cultures of bacteria were grown in submerged conditions at a temperature of 15 and 26oC for 48 h. The synthetic and natural substrates such as p-nitrophenyl-glycosides, soluble starch, gelatin, casein and Elastin-Congo red were used to study the enzymatic activity of bacteria. Results. All studied strains showed α-fucosidase activity. Microbacterium foliorum, Sporosarcina aquimarina and Rothia sp. exhibited α-, β-xylosidase, β-glucosidase or β-N-acetylglucosaminidase activity in different ratios. That may indicate the presence of the enzymatic complex of hydrolysis of lichenan and xylan, which are part of polysaccharides of plants and lichens. P. fluorescens and M. foliorum also showed gelatinase activity. The enzymatic activity of bacteria was noted to be higher in the case of cultivating at 15oC compared to 26oC. The α-xylosidase of M. foliorum was optimally active at pH 6.0 and 35oC, the α-xylosidase of Rothia sp. – at pH 6.5 and 35oC. High level of stability was shown for these enzymes in the pH range from 4.0 to 7.0 and temperature from 15 to 35оС during 24 h. Conclusions. Antarctic lichens can be a source of bacterial producers of polysaccharide degrading enzymes with new properties and low temperature optimum. The Antarctic cold environment provides the great opportunities to study the adaptive mechanisms of microorganisms and their enzymatic systems in order to develop new biotechnologies.

Bacteriocinogenic Bacillus spp. Isolated from Korean Fermented Cabbage (Kimchi)—Beneficial or Hazardous?

Bacillus velezensis ST03 and ST32, Bacillus amyloliquefaciens ST06 and ST109, and Bacillus subtilis ST08 were isolated from artisanal-produced kimchi and were identified based on 16S rRNA partial sequencing. DNA obtained from the investigated bacilli generated positive results for lichenicidin, iturin, subtilosin, and surfactin on a strain-specific basis. The strains were found to produce antimicrobial metabolites with activity levels ranging between 800 and 1600 AU/mL on a strain-specific basis, as determined against Listeria monocytogenes ATCC15313. Moreover, all tested strains in this study were still active after treatment with proteolytic enzymes, even with reduced inhibition zones compared to the controls, pointing to additional antimicrobial activity possibly related to a non-proteinaceous molecular structure. Most probably these strains may express surfactin as an additional factor in their complex antimicrobial activity. B. amyloliquefaciens ST09 and B. velezensis ST03 and ST32 were characterized as positive for β-hemolysis. B. subtilis ST08 was shown to be positive for hblC and nheC and B. amyloliquefaciens ST109 for nheB. B. amyloliquefaciens ST109 generated positive results for gelatinase activity. The ability of the studied Bacillus strains to metabolize different carbohydrate sources was done based on the API50CHB test, while the enzyme production profile was recorded by the APIZym kit. All studied strains were positive producers for biogenic amines production. Studied Bacillus spp. strains were resistant to some of the evaluated antibiotics, tested according to recommendations of CLSI and EFSA.

Cajanus Cajan (l.) Millsp Aqueous Extracts against Melanoma Cell Line and their Proteases

Aims: Extract proteins with protease inhibitor (PI) activity from fresh organs of Cajanus cajan, using aqueous systems; study the activity of melanoma secreted proteases; investigate inhibitory effect of C. cajan extracts on melanoma proteases; and evaluated the effect of the extract, with the most protease inhibitor activity, on melanoma cell line (SK-MEL-28) viability. Material and Methods: Extracts of C. cajan leaves, stems, and roots were prepared using different aqueous systems. Protein content was evaluated by Bradford method, protein profile by gel electrophoresis by Laemmli method and, extracts PI activity against trypsin, papain, and pepsin. Melanoma cell line was cultured in Dulbecco's medium, and secreted proteases was obtained from culture supernatant. Characterization of melanoma proteases included substrates activity, optimum pH, and effect of specific PIs and cations on protease activity. Anticancer activity was investigated using C. cajan extracts (containing PIs) and SK-MEL-28 extracellular fraction (containing proteases). Cytotoxic effect of extract was assayed on SK-MEL-28 cells line using methylthiazol tetrazolium method. CC-P was submitted to thin layer chromatography to identify alkaloids, coumarins, flavonoids, saponins and terpenes. Results: C. cajan extracts showed different protein contents and protein profiles in electrophoresis analysis. C. cajan organs presented PIs activities against serine, cysteine, and aspartic proteases. Leaf extract prepared using phosphate buffer (CC-P) and stems extract prepared with water (CC-CA) had the best inhibitory activities against trypsin (~58%). Pepsin was the lowest inhibited (11-29%) and papain was the most inhibited (14-100%). Protease activity of melanoma fraction was the highest using casein as substrate, and two proteins with 150 and 100 kDa with gelatinase activity. These proteases has maximal activity at pH 7.0 and 9.0, and was importantly inhibited by benzamidine, 1,10-phenanthroline and EDTA, suggesting that serine and metalloproteases are secreted by SK-MEL-28 cells. CC-P was the most important inhibitor of melanoma proteases, and induced cytotoxicity on SK-MEL-28 cells in culture. Although there is correlation between melanoma protease inhibition and cell death, CC-P has secondary metabolites, as coumarins, flavonoids and terpenes that can have synergy of antitumor activity. Conclusion: C. cajan extracts have serine, cysteine, and aspartic protease inhibitor activities. CC-P had the best inhibition on melanoma proteases and it was also cytotoxic to melanoma cell in culture. Therefore, these PIs can be important strategy for cancer treatment because tumor cells secrete proteases that are crucial to cancer progression.

Single nucleotide polymorphisms in matrix metalloproteinase 2 (MMP2) enhance BRAFV600E mutation-mediated oncogenicity and invasiveness of papillary thyroid cancer cells

Thyroid cancer is a common endocrine neoplasm. Despite its good prognosis, it can lead to significant mortality due to metastasis and recurrence. However, the factors involved in metastasis are not well studied. Therefore, we selected matrix metalloproteinases 2 (MMP2) and determined whether it has any role in thyroid cancer. We sequenced the exons of MMP2 in 211 samples including 16 multi-nodular goiters and 195 differentiated thyroid cancers. We identified four non-synonymous single nucleotide polymorphisms (SNPs) of the MMP2 gene in 3.06% (6/195) thyroid cancers. Of the 4 MMP2 SNPs, 3 (75%) concomitantly had BRAFV600E. Hence, we speculated that the MMP2 SNPs may likely cooperate with BRAFV600E in promoting tumor aggressiveness. Overexpression of two MMP2 SNPs (P38L and T458I) exhibited markedly enhanced gelatinase activity with an intact dimerization and induced strong cortactin foci formation in HEK293T cells. Stable expression of the two MMP2 SNPs in BRAFV600E positive BCPAP cells dramatically enhanced cell proliferation, colony formation, and focus formation. Analysis of the morphology of MMP2 SNP bearing BCPAPV600E cells exhibited highly invasive phenotypes characterized by a high rate of wound healing and enhanced cell invasion compared with parental BCPAPV600E cells bearing vector. We also determined that BCPAPV600E cells stably transfected with MMP2 SNPs were highly sensitive to the treatment of BRAF inhibitor, PLX4720. Our study demonstrates that MMP2 SNPs could cooperate with BRAFV600E to promote oncogenicity, migration, and invasiveness of PTC cells. These results suggest that a subset of papillary thyroid cancer with this genetic makeup can benefit from BRAF-mediated therapeutic interventions.

Ileocolonic Healing After Extended Small Bowel Resection in Mice: NOD2 Deficiency Impairs Anastomotic Healing and Postoperative Outcome

Background Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) mutations are a genetic risk factor for Crohn disease. Ileocecal resection is the most often performed surgery in Crohn disease. We investigated the effect of Nod2 knockout (KO) status on anastomotic healing after extended ileocecal resection (ICR) in mice. Methods Male C57BL6/J wild-type and Nod2 KO mice underwent an 11 cm resection of the terminal ileum including the cecum. An end-to-end jejuno-colostomy was performed. Animals were killed after 5 days investigating bursting pressure, hydroxyproline content, and expression of matrix metabolism genes, key cytokines, and histology of the anastomosis. Results Mortality was higher in the Nod2 KO group but not because of local or septic complications. Bursting pressure was significantly reduced in the Nod2 KO mice (32.5 vs 78.0 mmHg, P < 0.0024), whereas hydroxyprolin content was equal. The amount of granulation tissue at the anastomosis was similar but more unstructured in the Nod2 KO mice. Gene expression measured by real-time polymerase chain reaction showed significantly increased expression for Collagen 1alpha and for collagen degradation as measured by matrix metalloproteinase-2, -9, and -13 in the Nod2 KO mice. Gelatinase activity from anastomotic tissue was enhanced by Nod2 status. Gene expression of arginase I, tumor necrosis factor-α, and transforming growth factor-ß but not inducible nitric oxide synthase were also increased at the anastomosis in the Nod2 KO mice compared with the control mice. Conclusions We found that Nod2 deficiency results in significantly reduced bursting pressure after ileocecal resection. This effect is mediated via an increased matrix turnover. Patients with genetic NOD2 variations may be prone to anastomotic failure after bowel resection.

Isolation, molecular characterization and preliminary screening for probiotic properties of lactobacillus fermentum from indigenous dahi

Indigenous dahi is an analogue to fermented milk product of Pakistan produce by uncharacterized strains of LAB. It hides lots of health beneficial properties and unexplored micro-flora. On microbial examination of indigenous dahi, it was found that L. fermentum was most dominant spp. (22 isolates) of LAB in it. Upon safety assessment eleven showed negative hemolytic and gelatinase activity. All these eleven strains were molecularly characterized through PCR by using universal primers (9F and 1510R). Obtained sequences were submitted to Gene data base of NCBI under accession numbers KX944639-42, KX957930-33 and KY069971-73. Furthermore these strains were screened on the basis of acid and bile tolerance and antimicrobial activity. KX957931, KX957932 and KY069973 were selected for further studies of functional attributes.