Disparate outer membrane exclusionary properties underlie intrinsic resistance to hydrophobic substances in Pseudomonas spp. isolated from surface waters under triclosan selection.

Representative members of surface water microbiota were obtained from three unrelated municipal sites in Oklahoma by direct plating under selection by the hydrophobic biocide triclosan. Multiple methods were employed to determine if intrinsic triclosan resistance reflected resistance to hydrophobic molecules by virtue of outer membrane impermeability. While all but one organism isolated in the absence of triclosan were able to initiate growth on MacConkey agar, only one was able to initiate significant growth with triclosan present. In contrast, all bacteria selected with triclosan were identified as Pseudomonas spp. using 16S RNA gene sequencing and exhibited growth comparable to Pseudomonas aeruginosa controls in the presence of hydrophobic antibacterial agents to include triclosan. Two representative bacteria isolated in the absence of triclosan allowed for greater outer membrane association with the fluorescent hydrophobic probe 1-N-phenylnapthylamine than did two triclosan-resistant isolates. Compound 48/80 disruption of outer membrane impermeability properties for hydrophobic substances either partially or fully sensitized nine of twelve intrinsically resistant isolates to triclosan. These data suggest that outer membrane exclusion underlies intrinsic resistance to triclosan in some, but not all Pseudomonas spp. isolated by selection from municipal surface waters and implicates the involvement of concomitant triclosan resistance mechanisms.

Probiotic interference of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 with the opportunistic fungal pathogen Candida albicans.

Candida albicans is the most important Candida species causing vulvovaginal candidiasis (VVC). VVC has significant medical and economical impact on women's health and wellbeing. While current antifungal treatment is reasonably effective, supportive and preventive measures such as application of probiotics are required to reduce the incidence of VVC. We investigated the potential of the probiotics Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 towards control of C. albicans. In vitro experiments demonstrated that lactic acid at low pH plays a major role in suppressing fungal growth. Viability staining following cocultures with lactobacilli revealed that C. albicans cells lost metabolic activity and eventually were killed. Transcriptome analyses showed increased expression of stress-related genes and lower expression of genes involved in fluconazole resistance, which might explain the increased eradication of Candida in a previous clinical study on conjoint probiotic therapy. Our results provide insights on the impact of probiotics on C. albicans survival.

Status of bacterial colonization, Toll-like receptor expression and nuclear factor-kappa B activation in normal and diseased human livers.

Epidemiological data on bacterial translocation (BT), colonization and inflammation in normal human livers is lacking. In this study we investigated the status of bacterial colonization and inflammation in the normal, cirrhotic primary biliary cirrhosis (PBC), and nonalcoholic steatohepatitis (NASH) human liver tissues. Comparatively normal livers showed increased bacterial colonization than PBC and NASH. We analyzed mRNA levels of Toll-like receptors (TLR) 2 and TLR4, and protein levels of TLR4. Phosphorylated IKKα (pIKKα) protein estimation served as a marker for nuclear factor-kappa B (NF-κB) activation. In spite of the increased bacterial colonization in normal liver tissues, lower levels of TLR2/4 mRNA and TLR4 and pIKKα proteins were found compared to PBC and NASH indicating the maintenance of suppressed inflammation and immune tolerance in normal livers. To our knowledge, this is the first clinical evidence showing suppressed inflammation despite bacterial colonization in normal human livers thus maintaining liver immune homeostasis.

Intestinal Microbiome and Metal Toxicity.

The human gut microbiome is considered critical for establishing and maintaining intestinal function and homeostasis throughout life. Evidence for bidirectional communication with the immune and nervous systems has spawned interest in the microbiome as a key factor for human and animal health. Consequently, appreciation of the microbiome as a target of xenobiotics, including environmental pollutants such as heavy metals, has risen steadily because disruption of a healthy microbiome (dysbiosis) has been linked to unfavorable health outcomes. Thus, toxicology must consider toxicant effects on the host's microbiome as an integral part of the holobiont. We discuss current findings on the impact of toxic metals on the composition, diversity, and function of the gut microbiome as well as the modulation of metal toxicity by the microbiome. Present limitations and future needs in elucidating microbiome-metal interactions and the potential of harnessing beneficial traits of the microbiota to counteract metal toxicity are also considered.

Role of sex steroid receptors in pathobiology of hepatocellular carcinoma.

The striking gender disparity observed in the incidence of hepatocellular carcinoma (HCC) suggests an important role of sex hormones in HCC pathogenesis. Though the studies began as early as in 1980s, the precise role of sex hormones and the significance of their receptors in HCC still remain poorly understood and perhaps contribute to current controversies about the potential use of hormonal therapy in HCC. A comprehensive review of the existing literature revealed several shortcomings associated with the studies on estrogen receptor (ER) and androgen receptor (AR) in normal liver and HCC. These shortcomings include the use of less sensitive receptor ligand binding assays and immunohistochemistry studies for ERalpha alone until 1996 when ERbeta isoform was identified. The animal models of HCC utilized for studies were primarily based on chemical-induced hepatocarcinogenesis with less similarity to virus-induced HCC pathogenesis. However, recent in vitro studies in hepatoma cells provide newer insights for hormonal regulation of key cellular processes including interaction of ER and AR with viral proteins. In light of the above facts, there is an urgent need for a detailed investigation of sex hormones and their receptors in normal liver and HCC. In this review, we systematically present the information currently available on androgens, estrogens and their receptors in normal liver and HCC obtained from in vitro, in vivo experimental models and clinical studies. This information will direct future basic and clinical research to bridge the gap in knowledge to explore the therapeutic potential of hormonal therapy in HCC.

Fecal microbiota in the female prairie vole (Microtus ochrogaster).

We examined the fecal microbiota of female prairie voles. This species is socially and, likely, sexually monogamous, and thus serves as a valuable model in which to examine the interaction between the microbiota-gut-brain axis and social behavior. At present, little is known about the gastrointestinal microbiota of prairie voles; therefore, we performed a first characterization of the fecal microbiota using 16S rRNA gene amplicon sequencing. Semiconductor sequencing technology on an Ion Torrent PGM platform was used to assess the composition of fecal microbiotas from twelve female prairie voles. Following quality filtering, 1,017,756 sequencing reads were classified from phylum to genus level. At the phylum level, Firmicutes, Bacteroidetes, and Saccharibacteria were the predominant taxa, while the Bacteriodales, Erysipelotrichaceae, Ruminococcaceae, and Lachnospiraceae contributed the most dominant microbial groups and genera. Microbial community membership was most similar between vole sibling pairs, but consideration of taxon abundances weakened these associations. The interdependence of host factors such as genetics and behavior with the gastrointestinal microbiota is likely to be particularly pronounced in prairie voles. Our pilot characterization of the prairie vole intestinal microbiota revealed a microbial community composition remarkably consistent with the monogastric alimentary system of these rodents and their diet rich in complex plant carbohydrates. The highly social nature of these animals poses specific challenges to microbiome analyses that nonetheless are valuable for advancing research on the microbiota-gut-brain-behavior axis. Our study provides an important basis for future microbiome research in this emerging model organism for studying social behavior.

Postnatal maturation of the intestinal epithelial barrier in prairie voles.

Intestinal epithelium develops during gestation and continues to mature post-natally into a selective barrier that will protect the individual while still allowing passage of nutrients. Until fully mature, the risk of translocation of microorganisms, toxins or antigens into the sub-epithelial tissue is high and could result in pathologies with life-altering consequences, or even premature death. Because of their monogamous mating system, prairie voles are an emerging model for studying the role of the intestinal microbiota in modulating social behavior via the microbiota-gut-brain-behavior axis. However, knowledge about the voles' intestinal barrier maturation is lacking. Understanding the maturation of the intestine epithelial barrier can complement the extensive behavioral literature for future studies involving the vole gut-brain axis. In this study, we characterized intestinal barrier function by demonstrating that two-week-old prairie voles have high paracellular absorption of FITC-dextran molecules prior to markedly decreased permeability at three weeks of age. In light of the fundamental role of tight junctions in maintaining epithelial integrity regulating intestinal permeability, we examined tight junction gene expression profiles. Transmission electron microscopy was used to visualize tight junction structure. Our results provide a timeline for intestinal barrier maturation and point to tight junction proteins involved in this process in prairie voles.

Lactobacilli with probiotic potential in the prairie vole (Microtus ochrogaster).

BACKGROUND: Recent research suggests integration of the intestinal microbiota in gut-brain communication which could lead to new approaches to treat neurological disorders. The highly social prairie voles are an excellent model system to study the effects of environmental factors on social behavior. For future studies on the role of probiotics in ameliorating disorders with social withdrawal symptoms, we report the characterization of intestinal Lactobacillus isolates with probiotic potential from voles. METHODS AND RESULTS: 30 bacterial strains were isolated from the vole intestine and found to be distinct but closely related to Lactobacillus johnsonii using 16S rRNA gene sequencing and Random Amplification of Polymorphic DNA fingerprinting. In vitro characterizations including acid and bile tolerance, antimicrobial effects, antibiotic susceptibility, and adherence to intestinal epithelial cells were performed to assess the probiotic potential of selected strains. Since previous studies revealed that mercury ingestion triggers social deficits in voles, mercury resistance of the probiotic candidates was evaluated which could be an important factor in preventing/treating these behavioral changes. CONCLUSIONS: This study demonstrates that lactobacilli with probiotic potential are present in the vole intestine. The Lactobacillus isolates identified in this study will provide a basis for the investigation of probiotic effects in the vole behavioral model system.

The effect of hyaluronic acid on biofunctionality of gelatin-collagen intestine tissue engineering scaffolds.

The creation of engineered intestinal tissue has recently stimulated new endeavors with the ultimate goal of intestinal replacement for massive resections of bowel. In this context, we investigated the effect of hyaluronic acid (HA) on the physicochemical characteristics of gelatin-collagen scaffolds and its cytocompatibilty to the human intestinal epithelial Caco-2 cell line in vitro. Gelatin/collagen hybrid scaffolds with different concentrations of HA were prepared by solvent casting and freeze-drying techniques and subsequent chemical crosslinking by genipin. The morphologies of the scaffolds were characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. In vitro tests were carried out in phosphate-buffered saline (PBS) solution to study the swelling ratio and the biostability of the scaffolds. It was found that the porous structure of the scaffolds could be tailored by further addition of HA. Moreover, both the swelling ratio and the degradation rate of the scaffold increased by addition of HA. A resazurin-based cell viability assay was employed to determine the viability and estimate the number of scaffold-adherent Caco-2 cells. The assay indicated that the scaffolds were all cytocompatible. We concluded that addition of less than 15% HA to scaffolds with a composition of 9:1 gelatin:collagen results only in incremental improvement in the structural characteristics and cytocompatibility of the gelatin-collagen scaffolds. However, the scaffolds with 25% HA exhibited remarkable enhancement in physicochemical characteristics of the scaffolds including cell viability, growth, and attachment as well as their physical structure.

Green synthesis of a new gelatin-based antimicrobial scaffold for tissue engineering.

With the aim of developing appropriate scaffolds for tissue engineering to suppress the formation of biofilms, an effective one-pot process was applied in this study to produce scaffolds with inherent antibacterial activity. A new method to synthesize genipin-crosslinked gelatin/nanosilver scaffolds with "green" in situ formation of silver nanoparticles by heat treatment is presented in this paper. In this procedure, toxic solvents, reducing agents, and stabilizing agents are avoided. UV-visible absorption spectra of the synthesized gelatin/nanosilver solutions were obtained immediately and three months after the synthesis revealing the presence and high stability of the silver nanoparticles. The TEM of gelatin/nanosilver solutions showed silver particles with spherical shapes that were less than 5nm in size. Interestingly, contact angle was found to increase from 80° to 125° with the increase in concentration of nanosilver in gelatin. All gelatin/nanosilver solutions showed antimicrobial activity against Staphylococcus aureus and Escherichia coli. However, only the highest concentration showed antifungal effects against Candida albicans pathogens. Scaffolds were prepared by a lyophilization technique from this solution and their antimicrobial activities were examined. Introducing this facile green one-pot process of synthesizing scaffolds with antimicrobial and anti-biofilm properties may lead to key applications in tissue engineering techniques.