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.

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.

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.

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.

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.

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.

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.

Evaluation of the microbial flora found in woodwind and brass instruments and their potential to transmit diseases.

Previous studies of dental devices (toothbrushes, dentures, and protective athletic mouthguards) have demonstrated microbial contamination of these devices and possible transmission of infectious diseases to the users. Since woodwind and brass instruments come into intimate contact with the musician's oral cavity and often are passed from student to student without sanitization, the question arises as to whether these instruments are contaminated and can transmit microbial diseases. The purpose of this study was to determine if woodwind and brass instruments and/or their cases harbor opportunistic, pathogenic, or allergenic microorganisms that can be transmitted to the musician. The internal components of woodwind and brass instruments harbored opportunistic, pathogenic, and/or allergenic microorganisms. The highest concentrations of microorganisms were found consistently at the mouthpiece end, but there was evidence of contamination throughout the instruments and their cases. The close proximity of contaminated mouthpieces to the oral cavity could facilitate local and systemic dissemination of the resident opportunistic, pathogenic, and/or allergenic microorganisms. General dentists should determine whether patients play a brass or woodwind instrument and be aware of the possible impact of this activity on the oral cavity and the entire body.

Microbiota found in protective athletic mouthguards.

BACKGROUND: Protective athletic mouthguards (PAM) produce oral mucosal injuries and may be associated with other systemic conditions. HYPOTHESIS: With wear, PAM become contaminated by a range of microorganisms. The number of microorganisms in PAM can be reduced by daily use of an antimicrobial solution. STUDY TYPE: Case series. STUDY DESIGN AND METHODS: Sixty-two division I football player volunteers were divided into 4 groups, using PAM for varying lengths of time before surrendering them for microbial analysis. One group had their PAM soaked in an antimicrobial solution between uses. The PAM were analyzed qualitatively and quantitatively for bacteria, yeasts, and fungi, using previously accepted methods. RESULTS: The 62 football players surrendered a total of 81 PAM for microbial analysis. The PAM yielded 154 gram-positive cocci, 150 gram-positive bacilli, 21 gram-negative cocci, 31 gram-negative bacilli, 22 yeasts, and 107 molds. The most common species of gram-positive cocci were Staphylococcus spp. and Micrococcus spp. Only 3 PAM (4%) were positive for Staphylococcus aureus. The most common species of gram-positive bacilli were Brevibacterium spp. and Cellulomonas spp. The most common species of yeasts were Candida parapsilosis and Rhodotorula mucilaginosa, while the most common species of mold were Cochliobolus spp. and Penicillium chrysogenum. Soaking the PAM in an antimicrobial solution between uses substantially reduced the numbers of microorganisms. CONCLUSIONS: Substantial microbial contamination of PAM occurs with use. The microbial load can be reduced by soaking in an antimicrobial solution between uses. CLINICAL RELEVANCE: PAM are contaminated by microorganisms that have the potential to produce oral and systemic diseases. They should be sanitized daily and changed when they become sharp and/or jagged.

Substitution of methionine 63 or 83 in S100A9 and cysteine 42 in S100A8 abrogate the antifungal activities of S100A8/A9: potential role for oxidative regulation.

S100A8 and S100A9 and their heterocomplex calprotectin (S100A8/A9) are abundant cytosolic constituents in human neutrophils previously shown to possess antifungal activity. This study was designed to investigate mechanisms involved in the modulation of the antifungal properties of S100A8/A9. S100A8, S100A9 and site-directed mutants of both proteins were tested for their antifungal effect against Candida albicans in microplate dilution assays. Whereas S100A8 alone did not inhibit fungal growth, S100A9 by itself had a moderate antifungal effect. Combining both proteins had the strongest effect. Supporting a potential role for oxidation in S100A8/A9, substitution of methionine 63 or 83 of S100A9 resulted in the loss of antifungal activity. Additionally, the substitution to alanine of cysteine 42 of S100A8 also caused a loss of S100A8's ability to enhance S100A9's antifungal effect. Overall, our data indicate that both S100A8 and S100A9 are required for their fully active antifungal effect and that oxidation regulates S100A8/A9 antifungal activity through mechanisms that remain to be elucidated and evaluated. Finally, together with our previous work describing the oxidation-sensitive anti-inflammatory effects of S100A8/A9, we propose that S100A8/A9 exerts an anti-inflammatory activity in healthy state and that conditions associated with oxidative stress activate the antifungal activity of S100A8/A9.

Protective athletic mouthguards: do they cause harm?

BACKGROUND: Protective athletic mouthguards (PAMs) have been worn in competitive sports for more than 100 years. Today, participants in contact and noncontact sports wear PAMs. HYPOTHESIS: Wearing a PAM produces oral injury. STUDY TYPE: Case series. STUDY DESIGN AND METHODS: Sixty-two Division I football players voluntarily participated in the study. Before the beginning of the season, each player underwent a thorough oral examination, and all abnormal oral findings were photographed (hyperkeratosis, erythema, ulceration, and combinations thereof). At midseason, 14 players were given complete oral examinations, with all abnormal oral findings documented. At season end, all remaining players (n = 53) had complete oral examinations and photographs taken of abnormal oral findings. RESULTS: The preseason examination of 62 players found a total of 85 lesions (1.4 lesions per player) on the gingiva (n = 17), buccal mucosa (n = 60), and palate (n = 8). The 14 midseason players had 28 lesions (2.0 lesions per player) on gingiva (n = 8), buccal mucosa (n = 16), and tongue (n = 4). At season end, the 53 remaining players had 198 lesions (3.7 per player) on the gingiva (n = 96), buccal mucosa (n = 79), tongue (n = 18), and palate (n = 5). In addition, the lesion intensity scores progressively increased over the season. Because the palate did not come into direct contact with the PAM, it was used as an internal control. CONCLUSION: The wearing of a PAM may increase the number and intensity of oral mucosal injuries, which may cause localized soft tissue reactions such as hyperkeratosis, erythema, and ulceration. CLINICAL RELEVANCE: Because the PAM reduces tooth injury but may cause oral lesions, it should be sanitized daily and changed regularly and replaced whenever it becomes sharp and jagged or when the athlete develops an irritation in the mouth.

Phospholipase A2 and phospholipase B activities in fungi.

As saprophytes or disease causing microorganisms, fungi acquire nutrients from dead organic material or living host organisms. Lipids as structural components of cell membranes and storage compartments play an important role as energy-rich food source. In recent years, it also has become clear that lipids have a wide range of bioactive properties including signal transduction and cell to cell communication. Thus, it is not surprising that fungi possess a broad range of hydrolytic enzymes that attack neutral lipids and phospholipids. Especially during infection of a mammalian host, phospholipase A(2) (PLA(2)) enzymes released by fungi could play important roles not only for nutrient acquisition and tissue invasion, but for intricate modulation of the host's immune response. Sequencing of fungal genomes has revealed a wide range of genes encoding PLA(2) activities in fungi. We are just beginning to become aware of the significance these enzymes could have for the fungal cells and their interaction with the host.

Inactivation of the phospholipase B gene PLB5 in wild-type Candida albicans reduces cell-associated phospholipase A2 activity and attenuates virulence.

Phospholipases are critical for modification and redistribution of lipid substrates, membrane remodeling and microbial virulence. Among the many different classes of phospholipases, fungal phospholipase B (Plb) proteins show the broadest range of substrate specificity and hydrolytic activity, hydrolyzing acyl ester bonds in phospholipids and lysophospholipids and further catalyzing lysophospholipase-transacylase reactions. The genome of the opportunistic fungal pathogen Candida albicans encodes a PLB multigene family with five putative members; we present the first characterization of this group of potential virulence determinants. CaPLB5, the third member of this multigene family characterized herein is a putative secretory protein with a predicted GPI-anchor attachment site. Real-time RT-PCR gene expression analysis of CaPLB5 and the additional CaPLB gene family members revealed that filamentous growth and physiologically relevant environmental conditions are associated with increased PLB gene activity. The phenotypes expressed by null mutant and revertant strains of CaPLB5 indicate that this lipid hydrolase plays an important role for cell-associated phospholipase A(2) activity and in vivo organ colonization.

Selecting, testing and understanding probiotic microorganisms.

The interest in probiotics and the modulation of microbiota for restoring and maintaining health continues to gain momentum. Research is fueled by a need to develop alternatives to antibiotics and drugs that have severe side effects. It is recognised that bacteria play a major role in human and animal health, and how scientific advances help to explain how and when probiotics work. This minireview provides an update on critical studies, particularly since 2002, that are helping to explain the mechanisms of action of probiotic organisms.

The functional basis of mycophenolic acid resistance in Candida albicans IMP dehydrogenase.

Candida albicans is an important fungal pathogen of immunocompromised patients. In cell culture, C. albicans is sensitive to mycophenolic acid (MPA) and mizoribine, both natural product inhibitors of IMP dehydrogenase (IMPDH). These drugs have opposing interactions with the enzyme. MPA prevents formation of the closed enzyme conformation by binding to the same site as a mobile flap. In contrast, mizoribine monophosphate, the active metabolite of mizoribine, induces the closed conformation. Here, we report the characterization of IMPDH from wild-type and MPA-resistant strains of C. albicans. The wild-type enzyme displays significant differences from human IMPDHs, suggesting that selective inhibitors that could be novel antifungal agents may be developed. IMPDH from the MPA-resistant strain contains a single substitution (A251T) that is far from the MPA-binding site. The A251T variant was 4-fold less sensitive to MPA as expected. This substitution did not affect the k(cat) value, but did decrease the K(m) values for both substrates, so the mutant enzyme is more catalytically efficient as measured by the value of k(cat)/K(m). These simple criteria suggest that the A251T variant would be the evolutionarily superior enzyme. However, the A251T substitution caused the enzyme to be 40-fold more sensitive to mizoribine monophosphate. This result suggests that A251T stabilizes the closed conformation, and this hypothesis is supported by further inhibitor analysis. Likewise, the MPA-resistant strain was more sensitive to mizoribine in cell culture. These observations illustrate the evolutionary challenge posed by the gauntlet of chemical warfare at the microbial level.

Functional analysis of a vacuolar ABC transporter in wild-type Candida albicans reveals its involvement in virulence.

ATP-driven transport proteins belonging to the ATP-binding cassette (ABC) superfamily perform important functions in cell metabolism and detoxification. Compounds can be actively transported across membranes, including the plasma membrane or organellar membranes. The vacuole is an important organelle in fungal cells required for compartmentalization of metabolites as well as toxic substances. Sequestration into the vacuole is often energy-dependent. We present the first isolation and molecular analysis of a vacuolar ABC transporter gene in the opportunistic fungal pathogen Candida albicans. The protein encoded by the MLT1 gene is highly similar to Multiple Drug Resistance-associated Protein (MRP)-like transporters of yeast and higher organisms that form the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)/MRP subfamily of ABC transporters, a class of proteins so far not characterized in C. albicans. MLT1 expression is extensively growth phase-regulated, and gene transcripts are inducible by metabolic poisons. Gene replacement mutants generated in wild-type C. albicans with the dominant selection marker MPAR showed a profound reduction in virulence in a mouse peritonitis model that was reversed by complementation with an intact MLT1 gene. Hence, this report provides primary evidence for the involvement of vacuolar ABC transporters in fungal virulence.