Relatedness of Escherichia coli strains with different susceptibility phenotypes isolated from swine feces during ampicillin treatment.

The aim of this study was to examine the dynamics of the development of resistance in fecal Escherichia coli populations during treatment with ampicillin for 7 days in pigs. Before treatment, only 6% of the isolates were ampicillin resistant, whereas more than 90% of the isolates were resistant after days 4 and 7 of treatment. Ampicillin-resistant E. coli isolates were mainly multiresistant, and 53% of the isolates from the treated pigs had one phenotype that included resistance to six antibiotics (ampicillin, chloramphenicol, sulfonamides, tetracycline, trimethoprim, and streptomycin) at day 7. Determination of the frequency of the four phylogenetic groups showed that there was a shift in the E. coli population in ampicillin-treated pigs; before treatment 75% of the isolates belonged to phylogroup B1, whereas at day 7 85% of the isolates belonged to phylogroup A. Pulsed-field gel electrophoresis (PFGE) typing revealed that ampicillin treatment selected ampicillin-resistant isolates with genotypes which were present before treatment. Comparison of antimicrobial phenotypes and PFGE genotypes showed that resistance traits were disseminated by vertical transmission through defined strains. One PFGE genotype, associated with the six-antibiotic-resistant phenotype and including a specific combination of resistance determinants, was predominant among the ampicillin-resistant strains before treatment and during treatment. These data indicate that ampicillin administration selected various ampicillin-resistant isolates that were present in the digestive tract before any treatment and that E. coli isolates belonging to one specific PFGE genotype encoding resistance to six antibiotics became the predominant strains as soon as ampicillin was present in the digestive tract.

Impact of three ampicillin dosage regimens on selection of ampicillin resistance in Enterobacteriaceae and excretion of blaTEM genes in swine feces.

The aim of this study was to assess the impact of three ampicillin dosage regimens on ampicillin resistance among Enterobacteriaceae recovered from swine feces by use of phenotypic and genotypic approaches. Phenotypically, ampicillin resistance was determined from the percentage of resistant Enterobacteriaceae and MICs of Escherichia coli isolates. The pool of ampicillin resistance genes was also monitored by quantification of bla(TEM) genes, which code for the most frequently produced beta-lactamases in gram-negative bacteria, using a newly developed real-time PCR assay. Ampicillin was administered intramuscularly and orally to fed or fasted pigs for 7 days at 20 mg/kg of body weight. The average percentage of resistant Enterobacteriaceae before treatment was between 2.5% and 12%, and bla(TEM) gene quantities were below 10(7) copies/g of feces. By days 4 and 7, the percentage of resistant Enterobacteriaceae exceeded 50% in all treated groups, with some highly resistant strains (MIC of >256 microg/ml). In the control group, bla(TEM) gene quantities fluctuated between 10(4) and 10(6) copies/g of feces, whereas they fluctuated between 10(6) to 10(8) and 10(7) to 10(9) copies/g of feces for the intramuscular and oral routes, respectively. Whereas phenotypic evaluations did not discriminate among the three ampicillin dosage regimens, bla(TEM) gene quantification was able to differentiate between the effects of two routes of ampicillin administration. Our results suggest that fecal bla(TEM) gene quantification provides a sensitive tool to evaluate the impact of ampicillin administration on the selection of ampicillin resistance in the digestive microflora and its dissemination in the environment.

Time course of COX-1 and COX-2 expression during ischemia-reperfusion in rat skeletal muscle.

The aim of this study was to assess cyclooxygenase (COX)-1 and COX-2 expression in skeletal muscle after an ischemia-reperfusion (I/R). Male Sprague-Dawley rats were subjected to unilateral hindlimb ischemia for 2 h and then euthanized after 0, 1, 2, 4, 6, 10, 24, and 72 h of reperfusion. The COX protein and mRNA were assessed in control and injured gastrocnemius muscle. Muscle damage was indirectly determined by plasma creatine kinase activity and edema by weighing wet muscle. Creatine kinase activity in plasma increased as early as 1 h after reperfusion and returned to control levels by 72 h of reperfusion. Edema was observed at 6 and 10 h of reperfusion, but histological investigations showed an absence of tissular inflammatory cell infiltration. COX-1 mRNA was expressed in control muscle and was increased at 72 h of reperfusion, but the levels of associated COX-1 protein detected in control and injured gastrocnemius muscle were similar. COX-2 mRNA was not, or only slightly, detectable in control muscle and after I/R. In contrast, I/R induced major overexpression of COX-2 immunoreactivity at 6 and 10 h of reperfusion with a maximum at 10 h, whereas COX-2 protein was undetectable in control muscle. In conclusion, hindlimb I/R induced a large overexpression of COX-2 but not COX-1 protein between 6 and 10 h after injury. These results suggest a role for COX-2 enzyme in such pathophysiological conditions of the skeletal muscle.

Effect of experimental renal impairment on disposition of marbofloxacin and its metabolites in the dog.

The pharmacokinetics of marbofloxacin was studied in eight healthy female Beagle dogs before and after moderate renal impairment was induced experimentally. A single intravenous (i.v.) administration and repeated administration for 8 days (2 mg/kg, once-a-day) of marbofloxacin were studied. Renal impairment was induced by a right kidney nephrectomy and electrocoagulation of the left kidney. An increase (P < 0.001) in the plasma concentrations of urea (from 3.8+/-0.7 to 9.8+/-2.1 mmol/L) and creatinine (from 78.8+/-3.4 to 145.8+/-22.3 micromol/L), and a significant decrease (2.9+/-0.3 vs 1.5+/-0.2 mL/kg/min) (P < 0.001) in glomerular filtration rate were observed in the renal-impaired dogs. The clearance of marbofloxacin was slightly decreased after the induction of renal failure (1.6+/-0.2 to 1.4+/-0.1 mL/kg/min) (P < 0.05), but no significant variation of volume of distribution at steady state (Vss) and mean residence time (MRT) was observed after intravenous administration of marbofloxacin (P > 0.05). Following oral administration of marbofloxacin, an increase in total area under the concentration time curve (AUC) was observed after renal failure (from 10372+/-1710 to 11459+/-1119 mg x min/L) (P < 0.05), but indices of accumulation were not modified. An increase (P < 0.01) in the AUC of N-oxide-marbofloxacin was observed after surgery. In conclusion, renal impairment has no biologically relevant influence on marbofloxacin disposition and there is no need for dosage adjustment of marbofloxacin in dogs with mild renal impairment.

Neuropeptide FF receptors control morphine-induced analgesia in the parafascicular nucleus and the dorsal raphe nucleus.

The ability of (1DMe)Y8Fa (D.Tyr-Leu-(NMe)Phe-Gln-Pro-Gln-Arg-Phe-NH2), a selective neuropeptide FF analog resistant to enzymatic degradation, to control morphine-induced analgesia was investigated in rat after microinfusion into the dorsal raphe nucleus and the nucleus parafascicularis of the thalamus. Infusion of (1DMe)Y8Fa (2.5 nmol) in the nucleus raphe dorsalis did not modify the animal response in the tail-immersion test but significantly reversed analgesia induced by coinjected morphine (27 nmol). Similarly, (1DMe)Y8Fa (5 nmol) inhibited morphine effects in the hot-plate test after co-injection into the parafascicular nucleus. Furthermore, (1DMe)Y8Fa injected into the parafascicular nucleus attenuated analgesia induced by morphine injected into the nucleus raphe dorsalis and similarly, the neuropeptide FF analog in the nucleus raphe dorsalis decreased the effects of 27 nmol morphine injected in the parafascicular nucleus. The density of neuropeptide FF receptors did not decrease in the nucleus raphe dorsalis after lesion of serotonergic neurons by 5,7-dihydroxytryptamine. However, after this lesion, (1DMe)Y8Fa injected in the nucleus raphe dorsalis was no longer able to modify analgesic effects of morphine in hot-plate and tail-immersion tests. Similarly, the serotonin (5-HT) depletion induced by a systemic administration of para-chlorophenylalanine did not modify morphine analgesia microinjected into the nucleus raphe dorsalis and the parafascicular nucleus but blocked the ability of (1DMe)Y8Fa to reverse morphine effects in both nuclei. These data show that neuropeptide FF exerts anti-opioid effects directly into both the nucleus raphe dorsalis and the parafascicular nucleus and acts also at distance on opioid functions. Furthermore, anti-opioid effects of neuropeptide FF require functional serotonergic neurons although neuropeptide FF receptors are not carried on these neurons.

Neuropeptide FF receptors in rat brain: a quantitative light-microscopic autoradiographic study using [125I][D.Tyr1, (NMe)Phe3]NPFF.

The anatomical localization of neuropeptide FF receptors was examined by in vitro autoradiography techniques in rat brain sections by using [125I][D.Tyr1, (NMe)Phe3]NPFF. The specific binding of [125I][D.Tyr1, (NMe)Phe3]NPFF reached 90% of the total binding at 0.05 nM in rat spinal cord sections. Up to 40% of the specific binding of[125I][D.Tyr1, (NMe)Phe3]NPFF to rat spinal cord sections was still detectable following fixation with glutaraldehyde. Afterwards, the distribution of NPFF receptors was studied by light microscopy and their densities by microdensitometry with an image analysis system. In the light microscope, [125I][D.Tyr1, (NMe)Phe3]NPFF labelling appeared more or less uniformly distributed over nerve-cell bodies and surrounding neuropil. High concentrations of binding sites were detected in the presubiculum, parafascicular thalamic nucleus, gracile nucleus, spinal trigeminal tract nucleus, and a number of brainstem nuclei, with virtually no labelling in the cerebellum. In several areas a rostrocaudal gradient of sites concentration was observed. Neuropeptide FF receptors are well-placed to control incoming sensory and autonomic information processing. In contrast, the more recently developed areas of the forebrain possessed low density of sites. The distribution of [125I][D.Tyr1, (NMe)Phe3]NPFF binding sites should suggest anatomical substrates for the actions of neuropeptide FF.

Species differences in the localization of neuropeptide FF receptors in rodent and lagomorph brain and spinal cord.

Quantitative in vitro receptor autoradiography of [125I][D-Tyr1,(NMe)Phe3]NPFF was used to study the regional distribution of neuropeptide FF receptors in rodent and lagomorph brain. In rat, mouse, rabbit, and Afghan pika [125I][D-Tyr1,(NMe)Phe3]NPFF binding sites were enriched in the superficial layers of dorsal horn of the spinal cord and in parabrachial nucleus, central gray matter, hypothalamus, and reunions thalamic nucleus. In other neuroanatomical regions, important species differences in NPFF receptor patterns are observed. In marked contrast, the brain and the spinal cord of the Octodon degus are devoid of NPFF receptors. The present study shows that in different species regional variations in brain NPFF receptor binding occur.

Effects of neuropeptide FF analogs on morphine analgesia in the nucleus raphe dorsalis.

The effect of microinfusion into the nucleus raphe dorsalis (DR) of neuropeptide FF (NPFF) analogs on the antinociceptive effects of morphine was evaluated in rats, using the tail-immersion test. infusion of morphine into the DR induced a dose-dependent analgesia significantly reversed by co-infusion of 2.5 nmol opioid antagonist, naloxone. Similarly, 2.5 nmol NPFF and (1DMe)Y8Fa(D-Tyr-Leu-(NMe)Phe-Gln-Pro-Gln-Arg-Phe-NH2) or (3D)Y8Fa(D-Tyr-D-Leu-D-Phe-Gln-Pro-Gln-Arg-Phe-NH2), two neuropeptide FF analogs, inhibited morphine analgesia, although these peptides had no effect on nociceptive thresholds. This anti-opioid effect is indirect since NPFF analogs displayed no significant affinity towards mu and delta opioid binding sites in the DR. After intracerebroventricular infusion, morphine produced the same degree of analgesia as that measured after infusion into the nucleus raphe dorsalis and both NPFF analogs reversed morphine antinociception. This result is the first direct evidence that neuropeptide FF may act on opioid system at the DR and that several nuclei are involved in the suppression of morphine-induced antinociception.