Metagenomic and metatranscriptomic analyses reveal that biobed systems can enrich for antibiotic resistance and genetic mobility genes.

Antibiotic resistance gene pollution in the environment has been identified as a potential contributor to the global issue of antibiotic resistance prevalence, creating a need to identify and characterize environmental reservoirs for antibiotic resistance genes. Because many polluted environments have been shown to contain elevated levels of antibiotic resistance genes, agriculturally based pesticide bioremediation systems called 'biobeds' could serve as environmental reservoirs for antibiotic resistance genes, although this has never been extensively explored. Metagenomic and metatranscriptomic analyses of an on-farm biobed system sampled before and after a season of pesticide use demonstrated that in situ pesticide applications applied to biobeds can enrich for multidrug, sulphonamide, aminoglycoside and beta-lactam resistance genes. Additionally, this study demonstrated an enrichment for genes associated with gene mobilization, such as genes involved in horizontal gene transfer and plasmid mobility, as well as transposons and integrases.

Platelet-derived TLT-1 is a prognostic indicator in ALI/ARDS and prevents tissue damage in the lungs in a mouse model.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) affect >200 000 individuals yearly with a 40% mortality rate. Although platelets are implicated in the progression of ALI/ARDS, their exact role remains undefined. Triggering receptor expressed in myeloid cells (TREM)-like transcript 1 (TLT-1) is found on platelets, binds fibrinogen, and mediates clot formation. We hypothesized that platelets use TLT-1 to manage the progression of ALI/ARDS. Here we retrospectively measure plasma levels of soluble TLT-1 (sTLT-1) from the ARDS Network clinical trial and show that patients whose sTLT-1 levels were >1200 pg/mL had nearly twice the mortality risk as those with <1200 pg/mL (P < .001). After correcting for confounding factors such as creatinine levels, Acute Physiology And Chronic Health Evaluation III scores, age, platelet counts, and ventilation volume, sTLT-1 remains significant, suggesting that sTLT-1 is an independent prognostic factor (P < .0001). These data point to a role for TLT-1 during the progression of ALI/ARDS. We use a murine lipopolysaccharide-induced ALI model and demonstrate increased alveolar bleeding, aberrant neutrophil transmigration and accumulation associated with decreased fibrinogen deposition, and increased pulmonary tissue damage in the absence of TLT-1. The loss of TLT-1 resulted in an increased proportion of platelet-neutrophil conjugates (43.73 ± 24.75% vs 8.92 ± 2.4% in wild-type mice), which correlated with increased neutrophil death. Infusion of sTLT-1 restores normal fibrinogen deposition and reduces pulmonary hemorrhage by 40% (P ≤ .001) and tissue damage by 25% (P ≤ .001) in vivo. Our findings suggest that TLT-1 uses fibrinogen to govern the transition between inflammation and hemostasis and facilitate controlled leukocyte transmigration during the progression of ARDS.

Identifying the core bacterial and fungal communities within four agricultural biobeds used for the treatment of pesticide rinsates.

AIMS: This study evaluated the variability of bacterial and fungal communities within unique pesticide remediation biobeds. METHODS AND RESULTS: Four biobeds receiving different applied pesticide rinsates, were sampled throughout an operational season to determine pesticide removal efficacy and microbial communities. Biomixture samples collected from different biobed depths, were subjected to Illumina sequencing of the 16S rRNA (bacteria) and ITS2 (fungi) genes. Pesticide removal rates for all biobeds averaged 99%, with microbial community analysis revealing biobeds shared 60-70% of the most abundant bacterial and fungal orders, respectively. Though biobed depth did not greatly impact microbial community profile or diversity, bacterial and fungal taxa profiles between biobeds notably diverge at levels of genera and OTU. Biobed bacterial communities exhibited greater diversity than fungal communities between and within all biobeds. CONCLUSIONS: Biobeds receiving variable pesticide rinsates share a 'core' microbial community, exhibiting greater bacterial diversity relative to fungal diversity. Pesticide exposure increased bacterial diversity throughout the biobeds, while fungal diversity was variable, meriting further understanding of fungicide application to biobed fungal community stability. SIGNIFICANCE AND IMPACT OF THE STUDY: Biobeds achieve high treatment efficacy of unique pesticide rinsates, regardless of differentiation of specific genera in response to specific compounds; supporting biobeds as a robust engineered system for pesticide rinsates bioremediation.

Reference intervals for stool calprotectin in preterm neonates and their utility for the diagnosis of necrotizing enterocolitis.

OBJECTIVE: Calprotectin is an antimicrobial protein found in stool when released by granulocytes. We sought to create stool calprotectin reference ranges in preterm neonates and to evaluate whether levels exceeding the upper reference interval are diagnostic for necrotizing enterocolitis (NEC). STUDY DESIGN: Stool calprotectin was measured in premature neonates without gastrointestinal pathology to create reference intervals. For comparison, levels from infants undergoing "rule out NEC" evaluations were plotted on these reference intervals. RESULTS: Stool calprotectin reference intervals were created according to gestational age at birth and corrected gestational age. Levels during "rule out NEC" evaluations were more often above the upper reference interval with NEC vs. those without NEC. CONCLUSIONS: Stools from preterm neonates have a higher range of calprotectin than stools from healthy term neonates. In evaluating preterm neonates for NEC with stool calprotectin, a calprotectin upper reference interval that incorporates corrected gestational age best predicts the diagnosis of NEC.

Successful implementation of an Enhanced Recovery After Surgery program shortens length of stay and improves postoperative pain, and bowel and bladder function after colorectal surgery.

BACKGROUND: Despite international data indicating that Enhanced Recovery After Surgery (ERAS) programs, which combine evidence-based perioperative strategies, expedite recovery after surgery, few centers have successfully adopted this approach within the U.S. We describe the implementation and efficacy of an ERAS program for colorectal abdominal surgery in a tertiary teaching center in the U.S. METHODS: We used a multi-modal and continuously evolving approach to implement an ERAS program among all patients undergoing colorectal abdominal surgery at a single hospital at the University of California, San Francisco. 279 patients who participated in the Enhanced Recovery after Surgery program were compared to 245 previous patients who underwent surgery prior to implementation of the program. Primary end points were length of stay and readmission rates. Secondary end points included postoperative pain scores, opioid consumption, postoperative nausea and vomiting, length of urinary catheterization, and time to first solid meal. RESULTS: ERAS decreased both median total hospital length of stay (6.4 to 4.4 days) and post-procedure length of stay (6.0 to 4.1 days). 30-day all-cause readmission rates decreased from 21 to 9.4 %. Pain scores improved on postoperative day 0 (3.2 to 2.1) and day 1 (3.2 to 2.6) despite decreased opioid. Median time to first solid meal decreased from 4.7 to 2.7 days and duration of urinary catheterization decreased from 74 to 46 h. Similar improvements were observed in all other secondary end points. CONCLUSIONS: These results confirm that a multidisciplinary, iterative, team-based approach is associated with a reduction in hospital stay and an acceleration in recovery without increasing readmission rates.

Elevated fecal calprotectin levels during necrotizing enterocolitis are associated with activated neutrophils extruding neutrophil extracellular traps.

OBJECTIVE: Neonates with necrotizing enterocolitis (NEC) have higher calprotectin levels in stool than do healthy neonates. However, it is not known whether high stool calprotectin at the onset of bowel symptoms identifies neonates who truly have NEC vs other bowel disorders. STUDY DESIGN: Neonates were eligible for this study when an x-ray was ordered to 'rule-out NEC'. Stool calprotectin was quantified at that time and in a follow-up stool. Each episode was later categorized as NEC or not NEC. The location of calprotectin in the bowel was determined by immunohistochemistry. RESULTS: Neonates with NEC had higher initial and follow-up stool calprotectin levels than did neonates without NEC. Calprotectin in bowel from neonates with NEC was within neutrophil extracellular traps (NETs). CONCLUSION: At the onset of signs concerning for NEC, fecal calprotectin is likely to be higher in neonates with NEC. Calprotectin in their stools is exported from neutrophils via NETs.

Baseline and storm event monitoring of Bacteroidales marker concentrations and enteric pathogen presence in a rural Canadian watershed.

Bacteroidales 16S rRNA gene markers were evaluated for their use as a microbial source tracking tool in a well characterized 750 ha agricultural watershed in Nova Scotia, Canada. Water quality monitoring was conducted following the validation of host-specific and universal Bacteroidales (AllBac) markers for their proficiency in this particular geographic region, which provided further evidence that these markers are geographically stable. Increasing Escherichia coli concentrations were positively correlated (p < 0.01) with concentrations of the AllBac marker in water samples, suggesting that this universal marker is more suited as a positive DNA control rather than as an indicator of recent fecal contamination. Ruminant (BacR) and bovine (CowM2) specific marker detection was associated with increased runoff due to precipitation in sub-watersheds putatively impacted by cattle farming, demonstrating that the BacR and CowM2 markers can be used to detect the recent introduction of fecal matter from cattle farming activities during rainfall events. However, the human associated marker (BacH) was only detected once in spite of numerous on-site residential wastewater treatment systems in the watershed, suggesting that this assay is not sensitive enough to detect this type of human sewage source. E. coli O157:H7 and Salmonella spp. DNA was not detected in any of the 149 watershed samples; however, 114 (76.5%) of those samples tested positive for Campylobacter spp. No significant correlation (p > 0.05) was found between Campylobacter spp. presence and either E. coli or AllBac marker levels. Further studies should be conducted to assess the origins of Campylobacter spp. in these types of watersheds, and to quantify pathogen cell numbers to allow for a human health risk assessment.

Characterization of a mobile and multiple resistance plasmid isolated from swine manure and its detection in soil after manure application.

AIMS: To isolate and characterize multiple antibiotic resistance plasmids found in swine manure and test for plasmid-associated genetic markers in soil following manure application to an agricultural field. METHODS AND RESULTS: Plasmids were isolated from an erythromycin enrichment culture that used liquid swine manure as an inoculant. Plasmids were transformed into Escherichia coli DH10ß for subsequent characterization. We isolated and DNA sequenced a 22 102-bp plasmid (pMC2) that confers macrolide, and tetracycline resistances, and carries genes predicted to code for mercury and chromium resistance. Conjugation experiments using an pRP4 derivative as a helper plasmid confirm that pMC2 has a functional mobilization unit. PCR was used to detect genetic elements found on pMC2 in DNA extracted from manure amended soil. CONCLUSIONS: The pMC2 plasmid has a tetracycline-resistant core and has acquired additional resistance genes by insertion of an accessory region (12 762 bp) containing macrolide, mercury and chromium resistance genes, which was inserted between the truncated DDE motifs within the Tn903/IS102 mobile element. SIGNIFICANCE AND IMPACT OF THE STUDY: Liquid swine manure used for manure spreading contains multiple antibiotic resistance plasmids that can be detected in soil following manure application.

Discrete change in volatile anesthetic sensitivity in mice with inactivated tandem pore potassium ion channel TRESK.

BACKGROUND: We investigated the role of tandem pore potassium ion channel (K2P) TRESK in neurobehavioral function and volatile anesthetic sensitivity in genetically modified mice. METHODS: Exon III of the mouse TRESK gene locus was deleted by homologous recombination using a targeting vector. The genotype of bred mice (wild type, knockout, or heterozygote) was determined using polymerase chain reaction. Morphologic and behavioral evaluations of TRESK knockout mice were compared with wild-type littermates. Sensitivity of bred mice to isoflurane, halothane, sevoflurane, and desflurane were studied by determining the minimum alveolar concentration preventing movement to tail clamping in 50% of each genotype. RESULTS: With the exception of decreased number of inactive periods and increased thermal pain sensitivity (20% decrease in latency with hot plate test), TRESK knockout mice had healthy development and behavior. TRESK knockout mice showed a statistically significant 8% increase in isoflurane minimum alveolar concentration compared with wild-type littermates. Sensitivity to other volatile anesthetics was not significantly different. Spontaneous mortality of TRESK knockout mice after initial anesthesia testing was nearly threefold higher than that of wild-type littermates. CONCLUSIONS: TRESK alone is not critical for baseline central nervous system function but may contribute to the action of volatile anesthetics. The inhomogeneous change in anesthetic sensitivity corroborates findings in other K2P knockout mice and supports the theory that the mechanism of volatile anesthetic action involves multiple targets. Although it was not shown in this study, a compensatory effect by other K2P channels may also contribute to these observations.

Anaphylaxis complicating graft reperfusion during orthotopic liver transplantation: a case report.

Hemodynamic instability may occur during liver transplantation especially following unclamping the portal vein. A period of hypotension (postreperfusion syndrome) is usually responsive to treatment with fluids, calcium, sodium bicarbonate, and vasoactive drugs, but if hypotension persists, other causes must be sought out. In this report, we present a case in which anaphylaxis, most likely due to a component of the University of Wisconsin preservation solution, occurred coincident with liver reperfusion and severely exacerbated reperfusion hemodynamic instability. To our knowledge, this is the first report of anaphylaxis at the time of reperfusion and may provide an explanation for cases of vasoplegic syndrome associated with graft reperfusion.

Quantitative real-time PCR assays for sensitive detection of Canada goose-specific fecal pollution in water sources.

Canada geese (Branta canadensis) are prevalent in North America and may contribute to fecal pollution of water systems where they congregate. This work provides two novel real-time PCR assays (CGOF1-Bac and CGOF2-Bac) allowing for the specific and sensitive detection of Bacteroides 16S rRNA gene markers present within Canada goose feces.

Regional expression of the anesthetic-activated potassium channel TRESK in the rat nervous system.

The two-pore-domain potassium (K(2P)) channels contribute to background (leak) potassium currents maintaining the resting membrane potential to play an important role in regulating neuronal excitability. As such they may contribute to nociception and the mechanism of action of volatile anesthetics. In the present study, we examined the protein expression pattern of the K(2P) channel TRESK in the rat central nervous system (CNS) and peripheral nervous system (PNS) by immunohistochemistry. The regional distribution expression pattern of TRESK has both similarities and significant differences from that of other K(2P) channels expressed in the CNS. TRESK expression is broadly found in the brain, spinal cord and dorsal root ganglia (DRG). TRESK expression is highest in important CNS structures, such as specific cortical layers, periaqueductal gray (PAG), granule cell layer of the cerebellum, and dorsal horn of the spinal cord. TRESK expression is also high in small and medium sized DRG neurons. These results provide an anatomic basis for identifying functional roles of TRESK in the rat nervous system.

Evaluation of host-specific Bacteroidales 16S rRNA gene markers as a complementary tool for detecting fecal pollution in a prairie watershed.

Our ability to identify and eliminate fecal contamination of water, now and in the future, is essential to reduce incidences of waterborne disease. Bacterial source tracking is a recently developed approach for identifying sources of fecal pollution. PCR primers designed by Bernhard and Field [Bernhard, A.E., Field, K.G., 2000a. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66(10), 4571-4574] and Dick et al. [Dick, L.K., Bernhard, A.E., Brodeur, T.J., Santo Domingo, J.W., Simpson, J.M., Walters, S.P., Field, K.G., 2005. Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification. Appl. Environ. Microbiol. 71(6), 3184-3191] for the detection of human (HF183), pig (PF163) and ruminant (CF128) specific Bacteroidales 16s rRNA genetic markers were tested for their suitability in detecting fecal pollution in Saskatchewan, Canada. The sensitivity and specificity of these primers were assessed by testing eight raw human sewage samples and 265 feces from 12 different species in Saskatchewan. The specificity of each primer set was > or =94%. The accuracy of HF183 and PF163 to distinguish between the different species was 100%, whereas CF128 cross-reacted with 22% of the pig feces. Occurrence of the host-specific Bacteroidales markers and the conventional indicator Escherichia coli in relation to several enteropathogens was investigated in 70 water samples collected from different sites along the Qu'Appelle River (Saskatchewan, Canada). Human and ruminant fecal markers were identified in 41 and 14% of the water samples, respectively, whereas the pig marker was never detected in the river water. The largest concentrations in E. coli counts were concomitant to the simultaneous detection of HF183 and CF128. Thermotolerant Campylobacter spp., Salmonella spp. and Shiga toxin genes (stx1 and stx2)-positive E. coli (STEC) were detected in 6, 7 and 63% of the water samples, respectively. However, none of the stx positive water samples were positive for the E. coli O157:H7 gene marker (uidA). Odds ratios analysis suggests that CF128 may be predictive for the presence of Salmonella spp. in the river investigated. None of the fecal indicators were able to confidently predict the presence of thermotolerant Campylobacter spp. and STEC.

Assessment of the microbial quality of irrigation water in a prairie watershed.

AIMS: To assess levels of faecal contamination in the Qu'Appelle River (Saskatchewan, Canada) and its suitability for irrigation, by using the Colilert-18/Quanti-Tray technology. METHODS AND RESULTS: Various sites located along the Qu'Appelle River were sampled weekly from May to August 2005-2007. A total of 594 freshwater samples were collected and analysed for enumeration of Escherichia coli using the Colilert-18. The false-positive rate for E. coli detection using Colilert-18 was at most 1.5%. Throughout the irrigation period (June to August), up to 85% of the water samples collected from one of the irrigation water-pumping sites exceeded the recommended limit of 100 CFU per 100 ml. Spikes in E. coli counts were generally concomitant with the sudden rise in river flows. A sub-sample of confirmed E. coli isolates were typed by randomly amplified polymorphic DNA (RAPD). RAPD analysis revealed a high degree of genetic diversity among E. coli isolates. A significant association between RAPD patterns and the month of E. coli isolation was demonstrated. CONCLUSIONS: Colilert-18 provides an effective means for assessing microbial quality of irrigation water. SIGNIFICANCE AND IMPACT OF THE STUDY: Qu'Appelle River is subject to variability of faecal contamination during irrigation times and monitoring throughout irrigation season is important for ensuring safe production practices.

Molecular biology of background K channels: insights from K(2P) knockout mice.

K(2P) channels are a family of cellular proteins that are essential for electrical signaling throughout the body. There are six K(2P) channel subfamilies, consisting of 15 distinct mammalian genes. K(2P) channels display a remarkable range of regulation by cellular, physical and pharmacologic agents, including protein kinases, intracellular Ca(2+), changes in internal and external pH, anesthetic agents, heat, stretch and membrane deformers. The molecular and cellular mechanisms underlying this regulation are complex and cooperate at many different levels. Recent research has provided strong evidence that the spatiotemporal-specific expression of K(2P) channels are determinants of physiologic selectivity and specificity. In recent years, knockout mice have been generated with inactivated K(2P) channel genes. These animals shed new light on the contribution of K(2P) channels to normal and abnormal physiology. In this review, we summarize the published data on these mice to broaden the understanding of the role of K(2P) channel activity.

Knockout of the gene encoding the K(2P) channel KCNK7 does not alter volatile anesthetic sensitivity.

The molecular site of action for volatile anesthetics remains unknown despite many years of study. Members of the K(2P) potassium channel family, whose currents are potentiated by volatile anesthetics have emerged as possible anesthetic targets. In fact, a mouse model in which the gene for TREK-1 (KCNK2) has been inactivated shows resistance to volatile anesthetics. In this study we tested whether inactivation of another member of this ion channel family, KCNK7, in a knockout mouse displayed altered sensitivity to the anesthetizing effect of volatile anesthetics. KCNK7 knockout mice were produced by standard gene inactivation methods. Heterozygous breeding pairs produced animals that were homozygous, heterozygous or wild-type for the inactivated gene. Knockout animals were tested for movement in response to noxious stimulus (tail clamp) under varying concentrations of isoflurane, halothane, and desflurane to define the minimum alveolar concentration (MAC) preventing movement. Mice homozygous for inactivated KCNK7 were viable and indistinguishable in weight, general development and behavior from heterozygotes or wild-type littermates. Knockout mice (KCNK7-/-) displayed no difference in MAC for the three volatile anesthetics compared to heterozygous (+/-) or wild-type (+/+) littermates. Because inactivation of KCNK7 does not alter MAC, KCNK7 may play only a minor role in normal CNS function or may have had its function compensated for by other inhibitory mechanisms. Additional studies with transgenic animals will help define the overall role of the K(2P) channels in normal neurophysiology and in volatile anesthetic mechanisms.

Chirality in anesthesia II: stereoselective modulation of ion channel function by secondary alcohol enantiomers.

Chirality has been proposed as a means for distinguishing relevant from irrelevant molecular targets of action, but the sensitivity and specificity of this test is unknown for volatile anesthetics. We applied enantiomers of two chiral anesthetic alcohols (2-butanol and 2-pentanol) that are enantioselective for the minimum alveolar concentration (MAC) preventing movement in 50% of animals and one (2-hexanol) that was not to frog oocytes. Each oocyte expressed one of three anesthetic-sensitive ion channels: a Twik-related-spinal cord K+ (TRESK) channel, a gamma-amino butyric acid type A (GABA(A)) receptor and an N-methyl-d-aspartate (NMDA) receptor. Using voltage-clamp techniques, we found that 2-butanol was not enantioselective for any channel (e.g., 16 mM 2-butanol R(-) and S(-) enantiomers decreased current through an NMDA receptors by 44% +/- 3% [mean +/- se] and 37% +/- 4%, respectively); 2-pentanol was enantioselective for one channel (the GABA(A) receptor, the enantiomers increasing current by 277% +/- 20% and 141% +/- 30%); 2-hexanol was enantioselective for both GABA(A) and NMDA receptors (e.g., decreasing current through the NMDA receptor by 19% +/- 3% and 43% +/- 5%). We calculated the sensitivity and specificity of chirality as a test of anesthetic relevance under two scenarios: 1) all three channels were relevant mediators of MAC and 2) no channel was a mediator of MAC. These sensitivities and specificities were poor because there is no consistent correspondence between receptor and whole animal results. We recommend that enantioselectivity not be used as a test of relevance for inhaled anesthetic targets.

The ventilatory stimulant doxapram inhibits TASK tandem pore (K2P) potassium channel function but does not affect minimum alveolar anesthetic concentration.

TWIK-related acid-sensitive K(+)-1 (TASK-1 [KCNK3]) and TASK-3 (KCNK9) are tandem pore (K(2P)) potassium (K) channel subunits expressed in carotid bodies and the brainstem. Acidic pH values and hypoxia inhibit TASK-1 and TASK-3 channel function, and halothane enhances this function. These channels have putative roles in ventilatory regulation and volatile anesthetic mechanisms. Doxapram stimulates ventilation through an effect on carotid bodies, and we hypothesized that stimulation might result from inhibition of TASK-1 or TASK-3 K channel function. To address this, we expressed TASK-1, TASK-3, TASK-1/TASK-3 heterodimeric, and TASK-1/TASK-3 chimeric K channels in Xenopus oocytes and studied the effects of doxapram on their function. Doxapram inhibited TASK-1 (half-maximal effective concentration [EC50], 410 nM), TASK-3 (EC50, 37 microM), and TASK-1/TASK-3 heterodimeric channel function (EC50, 9 microM). Chimera studies suggested that the carboxy terminus of TASK-1 is important for doxapram inhibition. Other K2P channels required significantly larger concentrations for inhibition. To test the role of TASK-1 and TASK-3 in halothane-induced immobility, the minimum alveolar anesthetic concentration for halothane was determined and found unchanged in rats receiving doxapram by IV infusion. Our data indicate that TASK-1 and TASK-3 do not play a role in mediating the immobility produced by halothane, although they are plausible molecular targets for the ventilatory effects of doxapram.

A new look at the respiratory stimulant doxapram.

A number of life-threatening clinical disorders may be amenable to treatment with a drug that can stimulate respiratory drive. These include acute respiratory failure secondary to chronic obstructive pulmonary disease, post-anesthetic respiratory depression, and apnea of prematurity. Doxapram has been available for over forty years for the treatment of these conditions and it has a low side effect profile compared to other available agents. Generally though, the use of doxapram has been limited to these clinical niches involving patients in the intensive care, post-anesthesia care and neonatal intensive care units. Recent basic science studies have made considerable progress in understanding the molecular mechanism of doxapram's respiratory stimulant action. Although it is unlikely that doxapram will undergo a clinical renaissance based on this new understanding, it represents a significant advance in our knowledge of the control of breathing.