Why are bacteria refractory to antimicrobials?

La incidència de la resistència als antibiòtics en bacteris patògens està augmentant. Aquesta resistència es pot aconseguir mitjançant tres rutes clares: amb la inactivació del medicament, amb la modificació de la diana (target) i amb la disminució de la concentració del medicament que arriba a la diana. Des de fa temps se sap que els mecanismes de resistència a antibiòtics específics es poden adquirir a través de mutacions en el genoma bacterià o mitjançant l'addició de més gens durant el trasllat horitzontal de gens. Recentment, també s'ha descobert la importància dels diferents estats fisiològics per a la supervivència dels bacteris en presència d'antibiòtics. Ara és aparent que els bacteris tenen complexos mecanismes de resistència intrínsecs que sovint no es detecten en les proves estàndards de sensibilitat que es fan als antibiòtics en els laboratoris clínics. Entre aquests mecanismes intrínsecs, és de suma importància el desenvolupament de la resistència en bacteris que es troben en agregats associats a superfícies o biopel·lícules.The incidence of antibiotic resistance in pathogenic bacteria is rising. Antibiotic resistance can be achieved via three distinct routes: inactivation of the drug, modification of the target of action, and decreasing the concentration of drug that can reach the target. It has long been recognized that specific antibiotic resistance mechanisms can be acquired through mutation of the bacterial genome or by the addition of genes through horizontal gene transfer. Recent attention has also brought to light the importance of different physiological states for the survival of bacteria in the presence of antibiotics. It is now apparent that bacteria have complex, intrinsic resistance mechanisms that oftentimes are not detected in the standard antibiotic sensitivity tests performed in clinical laboratories. Paramount among these intrinsic mechanisms is the development of resistance in bacteria found in surface-associated aggregates or biofilms

A study to determine the feasibility of placing trained mentally retarded individuals into horticultural jobs in the Philadelphia area

Call number: LD2668 .T4 1986 H63Master of ScienceHorticulture, Forestry, and Recreation Resource

Pseudomonas Aeruginosa Reduces Vx-809 Stimulated F508del-Cftr Chloride Secretion by Airway Epithelial Cells

Background: P. aeruginosa is an opportunistic pathogen that chronically infects the lungs of 85% of adult patients with Cystic Fibrosis (CF). Previously, we demonstrated that P. aeruginosa reduced wt-CFTR Cl secretion by airway epithelial cells. Recently, a new investigational drug VX-809 has been shown to increase F508del-CFTR Cl secretion in human bronchial epithelial (HBE) cells, and, in combination with VX-770, to increase FEV1 (forced expiratory volume in 1 second) by an average of 3-5% in CF patients homozygous for the F508del-CFTR mutation. We propose that P. aeruginosa infection of CF lungs reduces VX-809 + VX-770- stimulated F508del-CFTR Cl secretion, and thereby reduces the clinical efficacy of VX-809 + VX-770. Methods and Results: F508del-CFBE cells and primary cultures of CF-HBE cells (F508del/F508del) were exposed to VX-809 alone or a combination of VX-809 + VX-770 for 48 hours and the effect of P. aeruginosa on F508del-CFTR Cl secretion was measured in Ussing chambers. The effect of VX-809 on F508del-CFTR abundance was measured by cell surface biotinylation and western blot analysis. PAO1, PA14, PAK and 6 clinical isolates of P. aeruginosa (3 mucoid and 3 non-mucoid) significantly reduced drug stimulated F508del-CFTR Cl secretion, and plasma membrane F508del-CFTR. Conclusion: The observation that P. aeruginosa reduces VX-809 and VX-809 + VX-770 stimulated F508del CFTR Cl secretion may explain, in part, why VX-809 + VX-770 has modest efficacy in clinical trials

Farnesol Induces Hydrogen Peroxide Resistance in Candida albicans Yeast by Inhibiting the Ras-Cyclic AMP Signaling Pathway

Farnesol, a Candida albicans cell-cell signaling molecule that participates in the control of morphology, has an additional role in protection of the fungus against oxidative stress. In this report, we show that although farnesol induces the accumulation of intracellular reactive oxygen species (ROS), ROS generation is not necessary for the induction of catalase (Cat1)-mediated oxidative-stress resistance. Two antioxidants, α-tocopherol and, to a lesser extent, ascorbic acid effectively reduced intracellular ROS generation by farnesol but did not alter farnesol-induced oxidative-stress resistance. Farnesol inhibits the Ras1-adenylate cyclase (Cyr1) signaling pathway to achieve its effects on morphology under hypha-inducing conditions, and we demonstrate that farnesol induces oxidative-stress resistance by a similar mechanism. Strains lacking either Ras1 or Cyr1 no longer exhibited increased protection against hydrogen peroxide upon preincubation with farnesol. While we also observed the previously reported increase in the phosphorylation level of Hog1, a known regulator of oxidative-stress resistance, in the presence of farnesol, the hog1/hog1 mutant did not differ from wild-type strains in terms of farnesol-induced oxidative-stress resistance. Analysis of Hog1 levels and its phosphorylation states in different mutant backgrounds indicated that mutation of the components of the Ras1-adenylate cyclase pathway was sufficient to cause an increase of Hog1 phosphorylation even in the absence of farnesol or other exogenous sources of oxidative stress. This finding indicates the presence of unknown links between these signaling pathways. Our results suggest that farnesol effects on the Ras-adenylate cyclase cascade are responsible for many of the observed activities of this fungal signaling molecule

Farnesol and Cyclic AMP Signaling Effects on the Hypha-to-Yeast Transition in Candida Albicans

Candida albicans, a fungal pathogen of humans, regulates its morphology in response to many environmental cues and this morphological plasticity contributes to virulence. Farnesol, an autoregulatory molecule produced by C. albicans, inhibits the induction of hyphal growth by inhibiting adenylate cyclase (Cyr1). The role of farnesol and Cyr1 in controlling the maintenance of hyphal growth has been less clear. Here, we demonstrate that preformed hyphae transition to growth as yeast in response to farnesol and that strains with increased cyclic AMP (cAMP) signaling exhibit more resistance to farnesol. Exogenous farnesol did not induce the hypha-to-yeast transition in mutants lacking the Tup1 or Nrg1 transcriptional repressors in embedded conditions. Although body temperature is not required for embedded hyphal growth, we found that the effect of farnesol on the hypha-to-yeast transition varies inversely with temperature. Our model of Cyr1 activity being required for filamentation is also supported by our liquid assay data, which show increased yeast formation when preformed filaments are treated with farnesol. Together, these data suggest that farnesol can modulate morphology in preformed hyphal cells and that the repression of hyphal growth maintenance likely occurs through the inhibition of cAMP signaling

Links between Anr and Quorum Sensing in Pseudomonas aeruginosa Biofilms

In Pseudomonas aeruginosa, the transcription factor Anr controls the cellular response to low oxygen or anoxia. Anr activity is high in oxygen-limited environments, including biofilms and populations associated with chronic infections, and Anr is necessary for persistence in a model of pulmonary infection. In this study, we characterized the Anr regulon in biofilm-grown cells at 1% oxygen in the laboratory strain PAO1 and in a quorum sensing (QS)-deficient clinical isolate, J215. As expected, transcripts related to denitrification, arginine fermentation, high-affinity cytochrome oxidases, and CupA fimbriae were lower in the Δanr derivatives. In addition, we observed that transcripts associated with quorum sensing regulation, iron acquisition and storage, type VI secretion, and the catabolism of aromatic compounds were also differentially expressed in the Δanr strains. Prior reports have shown that quorum sensing-defective mutants have higher levels of denitrification, and we found that multiple Anr-regulated processes, including denitrification, were strongly inversely proportional to quorum sensing in both transcriptional and protein-based assays. We also found that in LasR-defective strains but not their LasR-intact counterparts, Anr regulated the production of the 4-hydroxy-2-alkylquinolines, which play roles in quorum sensing and interspecies interactions. These data show that Anr was required for the expression of important metabolic pathways in low-oxygen biofilms, and they reveal an expanded and compensatory role for Anr in the regulation of virulence-related genes in quorum sensing mutants, such as those commonly isolated from infections

Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries

Background: Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions. Results: Through microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions. Conclusion: Our results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important neurotoxin that affects human health as well as ecosystem function.Plymouth State University Graduate Programs OfficeWoods Hole Oceanographic Institution Academic Programs OfficeNew Hampshire IDeA Network of Biological Research Excellence (NH-INBRE)National Center for Research Resources (U.S.) (Grant 5P20RR030360-03)National Institute of General Medical Sciences (U.S.) (Grant 8P20GM103506-03

Signaling through Lrg1, Rho1 and Pkc1 Governs Candida albicans Morphogenesis in Response to Diverse Cues

The capacity to transition between distinct morphological forms is a key virulence trait for diverse fungal pathogens. A poignant example of a leading opportunistic fungal pathogen of humans for which an environmentally responsive developmental program underpins virulence is Candida albicans. C. albicans mutants that are defective in the transition between yeast and filamentous forms typically have reduced virulence. Although many positive regulators of C. albicans filamentation have been defined, there are fewer negative regulators that have been implicated in repression of filamentation in the absence of inducing cues. To discover novel negative regulators of filamentation, we screened a collection of 1,248 C. albicans homozygous transposon insertion mutants to identify those that were filamentous in the absence of inducing cues. We identified the Rho1 GAP Lrg1, which represses filamentous growth by stimulating Rho1 GTPase activity and converting Rho1 to its inactive, GDP-bound form. Deletion of LRG1or introduction of a RHO1 mutation that locks Rho1 in constitutively active, GTP-bound state, leads to filamentation in the absence of inducing cues. Deletion of the Rho1 downstream effector PKC1 results in defective filamentation in response to diverse host-relevant inducing cues, including serum. We further established that Pkc1 is not required to sense filament-inducing cues, but its kinase activity is critical for the initiation of filamentous growth. Our genetic analyses revealed that Pkc1 regulates filamentation independent of the canonical MAP kinase cascade. Further, although Ras1 activation is not impaired in a pkc1Δ/pkc1Δ mutant, adenylyl cyclase activity is reduced, consistent with a model in which Pkc1 functions in parallel with Ras1 in regulating Cyr1 activation. Thus, our findings delineate a signaling pathway comprised of Lrg1, Rho1 and Pkc1 with a core role in C. albicans morphogenesis, and illuminate functional relationships that govern activation of a central transducer of signals that control environmental response and virulence programs

Using DTAGs to understand sound use, behavior, and vessel and associated noise effects in Southern Resident killer whales

Prey availability and disturbance from vessels and noise are identified threats to the recovery of endangered Southern Resident killer whales. Vessels and noise can mask echolocation signals used to capture fish prey and/or disrupt foraging behavior with implications for energy acquisition. In the U.S., vessel regulations have been implemented since 2011 to protect killer whales from vessel disturbance, particularly given the extent of whale-watching activities in the Salish Sea. We utilized suction cup-attached digital acoustic recording tags (DTAGs), consisting of hydrophones and movement sensors, to measure received noise levels, understanding killer whale use of sound, and determine effects of vessels and noise on subsurface behavior. During the 29 tag deployments on individually identified killer whales, we collected detailed geo-referenced vessel data concurrently as conditions allowed, along with opportunistic observations of predation to validate feeding. Received noise levels (dB re 1microPa) were significantly different across years but not consistently lower after the implementation of vessel regulations. Of the vessel factors considered, both vessel count and speed, but not distance, explained differences in noise levels, which may reflect changes in whale-watching vessel practices after regulations implementation. Additionally, the analysis of data from these animal-borne tags allow us to better understand subsurface foraging behavior involving the use of sound, to quantify foraging rates at an individual level, and to understand detailed vessel and noise effects. The results, along with those of other related studies, inform conservation and management measures that aim to promote Southern Resident recovery