Antimicrobial Use and Resistance in Swine Waste Treatment Systems

Chlortetracycline and the macrolide tylosin were identified as commonly used antimicrobials for growth promotion and prophylaxis in swine production. Resistance to these antimicrobials was measured throughout the waste treatment processes at five swine farms by culture-based and molecular methods. Conventional farm samples had the highest levels of resistance with both culture-based and molecular methods and had similar levels of resistance despite differences in antimicrobial usage. The levels of resistance in organic farm samples, where no antimicrobials were used, were very low by a culture-based method targeting fecal streptococci. However, when the same samples were analyzed with a molecular method detecting methylation of a specific nucleotide in the 23S rRNA that results in resistance to macrolides, lincosamides, and streptogramin B (MLS_B), an unexpectedly high level of resistant rRNA (approximately 50%) was observed, suggesting that the fecal streptococci were not an appropriate target group to evaluate resistance in the overall microbial community and that background levels of MLS_B resistance may be substantial. All of the feed samples tested, including those from the organic farm, contained tetracycline resistance genes. Generally, the same tetracycline resistance genes and frequency of detection were found in the manure and lagoon samples for each commercial farm. The levels of tetracycline and MLS_B resistance remained high throughout the waste treatment systems, suggesting that the potential impact of land application of treated wastes and waste treatment by-products on environmental levels of resistance should be investigated further.     

FAS inhibitor cerulenin reduces food intake and melanocortin receptor gene expression without modulating the other (an)orexigenic neuropeptides in chickens

Cerulenin, a natural fatty acid synthase (FAS) inhibitor, and its synthetic analog C75 are hypothesized to alter the metabolism of neurons in the hypothalamus that regulate ingestive behavior to cause a profound decrease of food intake and an increase in metabolic rate, leading to body weight loss. The bulk of data exclusively originates from mammals (rodents); however, such effects are currently lacking in nonmammalian species. We have, therefore, addressed this issue in broiler chickens because this species is selected for high growth rate and high food intake and is prone to obesity. First, we demonstrate that FAS messenger and protein are expressed in the hypothalamus of chickens. FAS immunoreactivity was detected in a number of brain regions, including the nucleus paraventricularis magnocellularis and the nucleus infundibuli hypothalami, the avian equivalent of the mammalian arcuate nucleus, suggesting that FAS may be involved in the regulation of food intake. Second, we show that hypothalamic FAS gene expression was significantly (P < 0.05) decreased by overnight fasting similar to that in liver, indicating that hypothalamic FAS gene is regulated by energy status in chickens. Finally, to investigate the physiological consequences of in vivo inhibition of fatty acid synthesis on food intake, we administered cerulenin by intravenous injections (15 mg/kg) to 2-wk-old broiler chickens. Cerulenin administration significantly reduced food intake by 23 to 34% (P < 0.05 to P < 0.0001) and downregulated FAS and melanocortin receptors 1, 4, and 5 gene expression (P < 0.05). However, the known orexigenic (neuropeptide Y, agouti gene-related peptide, orexin, and orexin receptor) and anorexigenic (pro-opiomelanocortin and corticotropin-releasing hormone) neuropeptide mRNA levels remained unchanged after cerulenin treatment. These results suggest that the catabolic effect of cerulenin in chickens may be mediated through the melanocortin system rather than the other neuropeptides known to be involved in food intake regulation.     

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.     

Fracture risk and the use of a diuretic (indapamide sr) ± perindopril: a substudy of the Hypertension in the Very Elderly Trial (HYVET)

Background

Chronic pancreatitis is an inflammatory disease which is characterized by an irreversible conversion of pancreatic parenchyma to fibrous tissue. Beside obstructive jaundice and pseudocyst formation, about half of the patients need surgical intervention due to untreatable chronic pain during the course of the disease. In most of the patients with chronic pancreatitis, the head of the pancreas is the trigger of the chronic inflammatory process. Therefore, resection of pancreatic head tissue must be the central part of any surgical intervention. However, it is unclear to which extent the surgical procedure must be radical in order to obtain a favourable outcome for the patients.

Design

A single centre randomized controlled, superiority trial to compare two techniques of duodenum preserving pancreatic head resection. Sample size: 65 patients will be included and randomized intraoperatively. Eligibility criteria: All patients with chronic pancreatitis and indication for surgical resection and signed informed consent. Cumulative primary endpoint (hierarchical model): duration of surgical procedure, quality of life after one year, duration of intensive care unit stay, duration of hospital stay. Reference treatment: Resection of the pancreatic head with dissection of the pancreas from the portal vein and transsection of the gland (Beger procedure). Intervention: Partial Resection of the pancreatic head without transsection of the organ and visualization of the portal vein (Berne procedure). Duration: September 2003-October 2007.

Organisation/responsibility

The trial is conducted in compliance with the protocol and in accordance with the moral, ethical, regulatory and scientific principles governing clinical research as set out in the Declaration of Helsinki (1989) and the Good Clinical Practice guideline (GCP). The Center for Clinical Studies of the Department of Surgery Heidelberg is responsible for planning, conducting and final analysis of the trial.     

Glutamate levels and transport in cat (Felis catus) area 17 during cortical reorganization following binocular retinal lesions

Glutamate is known to play a crucial role in the topographic reorganization of visual cortex after the induction of binocular central retinal lesions. In this study we investigated the possible involvement of the glial high-affinity Na+/K+-dependent glutamate transporters in cortical plasticity using western blotting and intracortical microdialysis. Basal extracellular glutamate levels and the re-uptake activity for glutamate have been determined by comparing the extracellular glutamate concentration before and during the blockage of glutamate removal from the synaptic cleft with the potent transporter inhibitor l-trans-pyrrolidine-3,4-dicarboxylic acid. In cats with central retinal lesions we observed increased basal extracellular glutamate concentrations together with a decreased re-uptake activity in non-deprived, peripheral area 17, compared with the sensory-deprived, central cortex of the same animal as well as the topographically matching regions of area 17 in normal subjects. Western blotting experiments revealed a parallel decrease in the expression level of the glial glutamate transporter proteins GLT-1 and GLAST in non-deprived cortex compared with sensory-deprived cortex of lesion cats and the corresponding regions of area 17 of normal subjects. This study shows that partial sensory deprivation of the visual cortex affects the removal of glutamate from the synaptic cleft and implicates a role for glial-neuronal interactions in adult brain plasticity.     

Studies on the multi-enzyme complex of yeast fatty-acid synthetase. Reversible dissociation and isolation of two polypeptide chains.

1. The multi-enzyme complex of fatty acid synthetase, Mr 2300,000, was dissociated by acylation with dimethyl maleic anhydride under conditions which lead to an acylation of about 30% of the epsilon amino groups of lysine. The complete dissociation into the subunits alpha and beta is demonstrated by analytical ultracentrifugation as well as disc gel electrophoresis. 2. This dissociation is reversible. Hydrolysis of the resulting protein dicarboxylic acid monoamides under mildly acidic conditions leads to the unmodified subunits, which can be reconstituted to form a complex displaying about 60% of the original activity. 3. The subunits were isolated by sucrose-density-gradient centrifugation and studied for the different partial enzyme activities involved in long-chain fatty acid synthesis: malonyl, palmitoyl and acetyl transferase, enoyl reductase and dehydratase were shown to be exclusive functions of the beta chains of the complex, confirming a pentafunctional role of this subunit.     

Identity of malonyl and palmitoyl transferase of fatty acid synthetase from yeast. Functional interrelationships between the acyl transferases.

Functional interrelationships between the acyl transferases of yeast fatty acid synthetase were investigated. In binding assays with synthetase modified by 5,5'-dithiobis(2-nitrobenzoic acid), 4--5 malonyl transferase entities per multienzyme complex molecule could be titrated. In the presence of palmitoyl-CoA these malonyl transferases were found inaccessible to malonyl-CoA, whereas the acetyl transferases were reactive towards acetyl-CoA. Between four and five palmitoyl transferase entities per synthetase equivalent were found reactive towards palmitoyl-CoA, the palmitoyl binding being inhibited by malonyl-CoA. Following palmitoyl binding the acetyl transferases were found towards acetyl-CoA. Substrate model assays were consistent with these data. It is concluded that malonyl and palmitoyl transferases are closely coupled enzyme components of the multienzyme complex which are fairly independent of the acetyl transferase entities. The molecular basis for the observed coupling will be given in the following paper.     

Partial purification and characterization of a pyruvate dehydrogenase-complex-inactivating enzyme from rat liver.

An enzyme inactivating the pyruvate dehydrogenase complex (inactivase) was purified about 8000-fold from rat liver by differential centrifugation, acid extraction of a lysosomerich 25000 g pellet, acetone fractionation, and adsorption on calcium phosphate gel. By exclusion chromatography on Sephadex G-100 a molecular weight of 21 000 was estimated. The purified enzyme was most stable at pH 5.8 in potassium phosphate buffer, and at pH 4.5 in McIlvaine buffer. At high dilutions the enzyme was very labile and was remarkably stabilized by high salt concentrations. Enzyme activity is inhibited by native rat blood serum, iodoacetamide and leupeptin, but not by phenylmethanesulphonyl fluoride, suggesting that it belongs to the class of thiol proteinases. Among various enzymes tested, only 2-oxoglutarate dehydrogenase was attacked by the inactivase to a similar extent to the pyruvate dehydrogenase complex. Studies on the inactivation mechanism indicate that although the overall reaction is completely lost after treatment with inactivase, each individual step of the multienzyme complex retains full catalytic activity. As judged from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the transacetylase subunit appears to be degraded into several smaller fractions.     

New experiments of biotin enzymes.

The objects of structural studies on biotin-enzymes were acetyl CoA-carboxylase and pyruvate carboxylase of Saccharomyces cerevisiae and beta-methylcrotonyl CoA-carboxylase and acetyl CoA-carboxylase of Achromobacter IV S. It was found that these enzymes can be arranged in three groups. In the first group, as represented by acetyl CoA-carboxylase of Achromobacter, the active enzyme could be resolved in three types of functional components: (1) the biotin-carboxyl carrier protein, (2) the biotin carboxylase, and (3) the carboxyl transferase. In the second group, as represented by beta-methylcrotonyl CoA-carboxylase from Achromobacter only two types of polypeptides are present. The one carries the biotin carboxylase activity together with the biotin-carboxyl-carrier protein, the other one carries the carboxyl transferase activity. In this third group, as represented by the two enzymes of yeast, all three catalytic functions are incorporated in one multifunctional polypeptide chain. The evolution of the different enzymes is discussed. The animal tissues acetyl CoA-carboxylase is under metabolic control, as known from previous studies. It thus has to be expected that the levels of malonyl CoA in livers of rats in all states of depressed fatty acid synthesis are much lower than under normal conditions because the carboxylation of acetyl CoA is strongly reduced and cannot keep pace with the consumption of malonyl CoA by fatty acid synthetase. A new highly sensitive assay method for malonyl CoA was developed which uses tritiated NADPH and measures the incorporation of radioactivity into the fatty acids formed from malonyl CoA in the presence of purified fatty acid synthetase. The application of this method to liver extracts showed that the level of malonyl CoA which amounts to about 7 nmoles per gram of wet liver drops to less than 10% within a starvation period of 24 hr and even further if the starvation period is extended to 48 hr. A low malonyl CoA concentration is also found in the alloxan diabetic animals and in animals being fed a fatty diet after starvation. On the other hand, feeding a carbohydrate rich diet leads to malonyl CoA levels surpassing the levels found after feeding a balanced diet. These observations reconfirm the concept that fatty acid synthesis is principally regulated by the carboxylation of acetyl CoA.     

Effects of yeast proteinase A, proteinase B and carboxypeptidase Y on yeast phosphofructokinase.

Incubation of a crude yeast extract containing phosphofructokinase with proteinase A, proteinase B or carboxypeptidase Y gave the following results: Proteinase B and carboxypeptidase Y did not change the activity of phosphofructokinase during incubation. On the other hand, incubation with proteinase A resulted in a 40-100% activation; continued incubation, however, led to an inactivation of the enzyme. Addition of allosteric effectors did not change the activation or inactivation process. The activated phosphofructokinase was not changed with respect to pH optimum and ATP inhibition. Molecular weight determination of phosphofructokinase in crude extracts in the presence of inhibitors of proteinase A indicated a molecular weight of 700000. Without inhibitors of proteinase A, the molecular weight was determined to be 600 000, while after 40-100% activation by proteinase A, a molecular weight of 500 000 was obtained. The activity profile of proteinase A in density gradients indicated that this enzyme is bound to variety of cellular proteins.