Characterization of microbial communities in anaerobic bioreactors using molecular probes

The microbial community structure of twenty-one single-phase and one two-phase full-scale anaerobic sewage sludge digesters was evaluated using oligonucleotide probes complementary to conserved tracts of the 16S rRNAs of phylogenetically defined groups of methanogens and sulfate-reducing bacteria. These probe results were interpreted in combination with results from traditional chemical analyses and metabolic activity assays. It was determined that methanogens in healthy mesophilic, single-phase sewage sludge digesters accounted for approximately 8–12% of the total community and thatMethanosarcinales andMethanomicrobiales constituted the majority of the total methanogen population.Methanobacteriales andMethanococcales played a relatively minor role in the digesters. Phylogenetic groups of mesophilic, Gram-negative sulfate-reducing bacteria were consistently present at significant levels:Desulfovibrio andDesulfobulbus spp. were the dominant sulfate-reducing populations,Desulfobacter andDesulfobacterium spp. were present at lower levels, andDesulfosarcina, Desulfococcus, andDesulfobotulus spp. were absent. Sulfate reduction by one or more of these populations played a significant role in all digesters evaluated in this study. In addition, sulfate-reducing bacteria played a role in favoring methanogenesis by providing their substrates. The analysis of the two-phase digester indicated that true phase separation was not accomplished: significant levels of active methanogens were present in the first phase. It was determined that the dominant populations in the second phase were different from those in the single-phase digesters.     

Induction of UDP-Glucose:Salicylic Acid Glucosyltransferase Activity in Tobacco Mosaic Virus-Inoculated Tobacco (Nicotiana tabacum) Leaves

Salicylic acid (SA) is a putative signal that activates plant resistance to pathogens. SA levels increase systemically following the hypersensitive response produced by tobacco mosaic virus (TMV) inoculation of tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. The SA increase in the inoculated leaf coincided with the appearance of a [beta]-glucosidase-hydrolyzable SA conjugate identified as [beta]-O-D-glucosylsalicylic acid (GSA). SA and GSA accumulation in the TMV-inoculated leaf paralleled the increase in the activity of a UDP-glucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35) ([beta]-GTase) capable of converting SA to GSA. Healthy tissues had constitutive [beta]-GTase activity of 0.076 milliunits g-1 fresh weight. This activity started to increase 48 h after TMV inoculation, reaching its maximum (6.7-fold induction over the basal levels) 72 h after TMV inoculation. No significant GSA or elevated [beta]-Gtase activity could be detected in the healthy leaf immediately above the TMV-inoculated leaf. The effect of TMV inoculation on the [beta]-GTase and GSA accumulation could be duplicated by infiltrating tobacco leaf discs with SA at the levels naturally produced in TMV-inoculated leaves (2.7-27.0 [mu]g g-1 fresh weight). Pretreatment of leaf discs with the protein synthesis inhibitor cycloheximide inhibited the induction of [beta]-GTase by SA and prevented the formation of GSA. Of 12 analogs of SA tested, only 2,6-dihydroxybenzoic acid induced [beta]-GTase activity.     

A Human Anti-M2 Antibody Mediates Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Cytokine Secretion by Resting and Cytokine-Preactivated Natural Killer (NK) Cells

The highly conserved matrix protein 2 (M2) is a good candidate for the development of a broadly protective influenza vaccine that induces long-lasting immunity. In animal models, natural killer (NK) cells have been proposed to play an important role in the protection provided by M2-based vaccines through a mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated the ability of the human anti-M2 Ab1-10 monoclonal antibody (mAb) to activate human NK cells. They mediated ADCC against M2-expressing cells in the presence of Ab1-10 mAb. Furthermore, NK cell pro-inflammatory cytokine and chemokine secretion is also enhanced when Ab1-10 mAb is present. We also generated cytokine-preactivated NK cells and showed that they still displayed increased effector functions in the presence of Ab1-10 mAb. Thus, our study has demonstrated that human resting and cytokine-preactivated NK cells may have a very important role in the protection provided by anti-M2 Abs.     

Natural variation,differentiation, and genetic trade‐offs of ecophysiological traits in response to water limitation in Brachypodium distachyon and its descendent allotetraploid B. hybridum (Poaceae)

Differences in tolerance to water stress may underlie ecological divergence of closely related ploidy lineages. However, the mechanistic basis of physiological variation governing ecogeographical cytotype segregation is not well understood. Here, using Brachypodium distachyon and its derived allotetraploid B. hybridum as model, we test the hypothesis that, for heteroploid annuals, ecological divergence of polyploids in drier environments is based on trait differentiation enabling drought escape. We demonstrate that under water limitation allotetraploids maintain higher photosynthesis and stomatal conductance and show earlier flowering than diploids, concordant with a drought‐escape strategy to cope with water stress. Increased heterozygosity and greater genetic variability and plasticity of polyploids could confer a superior adaptive capability. Consistent with these predictions, we document (1) greater standing within‐population genetic variation in water‐use efficiency (WUE) and flowering time in allotetraploids, and (2) the existence of (nonlinear) environmental clines in physiology across allotetraploid populations. Increased gas exchange and diminished WUE occurred at the driest end of the gradient, consistent with a drought‐escape strategy. Finally, we found that allotetraploids showed weaker genetic correlations than diploids congruous with the expectation of relaxed pleiotropic constraints in polyploids. Our results suggest evolutionary divergence of ecophysiological traits in each ploidy lineage.     

Design of a nutrient medium for bacteria of the genus Haemophilus--producers of restriction endonucleases

A culture medium for the cultivation of hemophilic bacteria, containing acidic casein hydrolysate, aminopeptide and fodder yeast extract, has been proposed. The growth-stimulating properties of this medium have been studied on 5 strains producing restrictases differing in their specificity. In growing these producer strains in a model ANKUM-2 fermenter with the supply of carbohydrate substrates (glucose, sucrose, glycerin) the yield of biomass, considered to be high for hemophilic bacteria (10-14 g of humid substance from 1 liter of the medium), has been achieved. As shown on H. influenzae Rc B-2297 used as an example, an increase in the yield of microbial biomass leads to a decrease in restrictase specific activity.     

Revisiting the ancient concept of botanical therapeutics

Mixtures of interacting compounds produced by plants may provide important combination therapies that simultaneously affect multiple pharmacological targets and provide clinical efficacy beyond the reach of single compound-based drugs. Developing innovative scientific methods for discovery, validation, characterization and standardization of these multicomponent botanical therapeutics is essential to their acceptance into mainstream medicine.     

Tools for the identification of bioactives impacting the metabolic syndrome: screening of a botanical extract library using subcutaneous and visceral human adipose-derived stem cell-based assays

Plant extracts continue to represent an untapped source of renewable therapeutic compounds for the treatment and prevention of illnesses including chronic metabolic disorders. With the increase in worldwide obesity and its related morbidities, the need for identifying safe and effective treatments is also rising. As such, use of primary human adipose-derived stem cells represents a physiologically relevant cell system to screen for bioactive agents in the prevention and treatment of obesity and its related complications. By using these cells in a primary screen, the risk and cost of identifying artifacts due to interspecies variation and immortalized cell lines is eliminated. We demonstrate that these cells can be formatted into 384-well high throughput screens to rapidly identify botanical extracts that affect lipogenesis and lipolysis. Additionally, counterscreening with human primary stem cells from distinct adipose depots can be routinely performed to identify tissue specific responses. In our study, over 500 botanical extracts were screened and 16 (2.7%) were found to affect lipogenesis and 4 (0.7%) affected lipolysis.     

Macrolide Resistance in Microorganisms at Antimicrobial-Free Swine Farms

To investigate the relationship between agricultural antimicrobial use and resistance, a variety of methods for quantification of macrolide-lincosamide-streptogramin B (MLS_B) resistance were applied to organic swine farm manure samples. Fluorescence in situ hybridization was used to indirectly quantify the specific rRNA methylation resulting in MLS_B resistance. Using this method, an unexpectedly high prevalence of ribosomal methylation and, hence, predicted MLS_B resistance was observed in manure samples from two swine finisher farms that reported no antimicrobial use (37.6% ± 6.3% and 40.5% ± 5.4%, respectively). A culture-based method targeting relatively abundant clostridia showed a lower but still unexpectedly high prevalence of resistance at both farms (27.7% ± 11.3% and 11.7% ± 8.6%, respectively), while the prevalence of resistance in cultured fecal streptococci was low at both farms (4.0%). These differences in the prevalence of resistance across microorganisms suggest the need for caution when extrapolating from data obtained with indicator organisms. A third antimicrobial-free swine farm, a breeder-to-finisher operation, had low levels of MLS_B resistance in manure samples with all methods used (<9%). Tetracycline antimicrobials were detected in manure samples from one of the finisher farms and may provide a partial explanation for the high level of MLS_B resistance. Taken together, these findings highlight the need for a more fundamental understanding of the relationship between antimicrobial use and the prevalence of antimicrobial resistance.Clinical data have documented a substantial rise in the levels of antimicrobial resistance (reviewed in reference 22). In response to this alarming rise, national and international initiatives have been developed to limit the use of antimicrobials in both human and veterinary medicine, with some successes. However, some of the data suggest a more complicated relationship between the patterns of antimicrobial use and the resulting prevalence of resistance. For both avoparcin and chloramphenicol, a ban was not effective in reducing the prevalence of resistance to the respective antimicrobial in pig isolates (2, 9). This may be due to coselection by the continued use of other types of antimicrobials (1, 15, 16, 33). Coselection by other antimicrobials, however, cannot explain the persistence of antimicrobial resistance for years after all use of antimicrobials was stopped, as documented in other studies of swine (13, 25). A better understanding of this complex relationship is needed to provide a basis for developing more-effective measures to control the prevalence of antimicrobial resistance. One means for investigating the factors influencing the prevalence of resistance is through comparisons between conventional farms and organic, antimicrobial-free farms (12, 13, 18, 25) or the wilderness (14, 19).The current study focused on macrolide antimicrobials, for which the most clinically relevant resistance mechanisms are efflux and target site modification (20). Resistance via modification of the target site on the ribosome may be achieved either through point mutations in rRNA or proteins or through acquisition of an erm gene catalyzing a site-specific mono- or dimethylation of the 23S rRNA (37). The point mutations confer various levels of resistance and degrees of cross-resistance (35), and their known distribution is currently limited, although this may simply reflect the historical experimental focus (20, 35). Dimethylation of A2058 (Escherichia coli numbering), on the other hand, consistently results in high-level resistance (for antimicrobial concentrations above 1 mg/ml) for three structurally unrelated classes of antimicrobials, macrolides, lincosamides, and streptogramin Bs, or macrolide-lincosamide-streptogramin B (MLS_B) antimicrobials, because of their shared target site (37). Constitutive expression of an erm dimethylase can also confer resistance to the newer ketolides, which are erythromycin (macrolide) derivatives developed for use on macrolide-resistant pathogens, and the degree of resistance correlates with the degree of methylation (11). The ribosomal methylation resistance mechanism is of particular concern for this work for the following three reasons. (i) It confers a high level of resistance. (ii) It can be acquired through horizontal gene transfer and thus has the potential for rapid spread. (iii) It is relevant to swine production environments in the United States because all three classes of MLS_B antimicrobials are used there. A variety of methods have been used to quantify macrolide resistance, including traditional culture-based methods (for an example, see reference 9), PCR (for examples, see references 27 and 32), or fluorescence in situ hybridization (FISH) (for an example, see reference 34) detection of specific point mutations known to result in resistance in the targeted microorganisms, using PCR to detect erm and mef (efflux) genes (for examples, see references 6 and 31) and using membrane hybridizations to detect the degree of methylation at A2058 (5, 18).In our previous study of swine production, a discrepancy was observed between culture-based measurements of resistance to the macrolide tylosin and membrane hybridizations quantifying the ribosomal methylation leading to MLS_B resistance (18). Cultured fecal streptococci showed a low prevalence of tylosin resistance (4.0%) in manure samples from an organic farm, as expected in the absence of the selective pressure imposed by the use of antimicrobials. However, membrane hybridizations quantifying the ribosomal methylation leading to MLS_B resistance in all bacteria in the swine waste samples suggested the presence of a much higher level of resistance (approximately 50%). One explanation for this discrepancy is that the prevalence of resistance in the fecal streptococci was not representative of the overall prevalence of resistance in this community. However, the high level of resistance measured with the molecular method was surprising in the absence of antimicrobial use and could also be explained as an artifact of the membrane hybridization methodology.The primary objectives of this paper were to resolve this discrepancy between culture-based and molecular methods and, if the unexpectedly high prevalence of antimicrobial resistance was confirmed, to investigate possible explanations. To accomplish the first objective, we developed a variation of FISH to indirectly quantify the specific rRNA methylation resulting in MLS_B resistance and provide insight into the identity of the putative resistant microorganisms. The major group identified, Clostridium cluster XIVa, was targeted with a culture-based method to provide an independent quantification of resistance. The results presented here have confirmed an unexpectedly high prevalence of MLS_B resistance at two organic farms. They also support the hypothesis that the prior discrepancy resulted from differences in the prevalence of resistance across groups of microorganisms.