The effect of Escherichia coli ZP strain with a conjugation-based colicin E7 delivery on growth performance, hematological, biochemical, and histological parameters, gut microbiota, and nonspecific immunity of broilers.

The Escherichia coli ZP strain (ZP) was constructed based on the known probiotic E. coli strain Nissle 1917. It was genetically modified to carry the colicin E7 synthesis gene encoding DNase on a conjugative plasmid and the colicin E7 immunity gene in the chromosome. The aim of this study was to evaluate the effects of the daily ZP per oral administration (5 × 108 or 5 × 1010 CFU per bird) on the growth performance, hematological, biochemical, histological parameters, gut microbiota, and nonspecific immunity of the 4-24 days old broilers. The ZP administration increased the abundance of genera Bacillus, Butyrivibrio, and Clostridium and did not influence the weight gain of 4-16 days old broilers. The biochemical parameters were within normal ranges for poultry in experimental and control groups. The ZP administration had no effect on the erythrocyte numbers, hemoglobin and immunoglobulin Y concentrations, but significantly increased the serum lysozyme concentration, leukocyte numbers, and reactive oxygen species production by phagocytes compared with the control group. It did not cause inflammatory changes in intestinal mucosa, Peyer's patches, and spleen. Thus, the ZP had no detrimental effects on broiler health and could be an efficient probiotic for the broiler colibacillosis prophylaxis.

Virulence potential of faecal Escherichia coli strains isolated from healthy cows and calves on farms in Perm Krai.

Cattle are a reservoir of pathogenic and potentially pathogenic Escherichia coli (E. coli) strains, which can pose a threat to human and animal health. The aim of the study was to evaluate the occurrence of 22 virulence-associated genes (VAGs), as well as the prevalence of antimicrobial drug resistance and three different bla-genes among 49 E. coli strains isolated from healthy cattle. The presence of VAGs that are common among diarrheagenic E. coli (DEC) strains and/or extraintestinal pathogenic E. coli (ExPEC) strains was determined by amplifying specific gene sequences by PCR. The following VAGs associated with DEC were found: east1 in 24.5 % of the studied E. coli strains, estI in 10.2 %, ehxA in 8.2 %, stx2 in 6.1 %, eltA in 4.1 %, estII and stx1 in 2.0 % of the studied strains. The prevalence of ExPEC VAGs was: fimH - 91.8 %, afa/draBC - 61.2 %, iutA - 44.9 %, flu - 32.7 %, sfaDE and hlyF - 30.6 %, iroN - 22.4 %, ompT and papC - 20.4 %, kpsMTII and hlyA - 18.4 %, iss - 14.3 %, usp - 2.0 %, cnf1 and iha were not detected among the studied strains. Based on the found co-occurrence of VAGs "classical", hetero-pathogenic and hybrid-pathogenic E. coli strains were found. E. coli strains isolated from cows had a higher diarrheagenic potential, whereas E. coli strains isolated from calves more frequently contained genes associated with the ExPEC pathotype. Among the studied E. coli strains, 77.6 % were resistant to ampicillin, 49.0 % to tetracycline, 20.4 % to chloramphenicol, 16.3 % to cefoperazone, 16.3 % to ceftriaxone, 16.3 % to aztreonam, 14.3 % to cefepime, 10.2 % to norfloxacin, 10.2 % to ciprofloxacin, 6.1 % to levofloxacin and 2.0 % to gentamicin. All strains were sensitive to meropenem and amikacin. 32.7 % of the studied E. coli strains were found to be multidrug resistant, as they were resistant to at least three groups of antibiotics. With PCR, the blaTEM, blaSHV, and blaCTX-M genes were detected in 100, 31.6, and 26.3 %, respectively, of strains resistant to at least one of the beta-lactam antibiotics. Thus, it was shown that the studied faecal E. coli of healthy cows and calves had a high hetero-pathogenic potential, therefore in the future molecular genetic characterization of these bacteria shall be an important part of the epizootic monitoring.

Estimation of the bacteriocin ColE7 conjugation-based "kill" - "anti-kill" antimicrobial system by real-time PCR, fluorescence staining and bioluminescence assays.

The efficiency of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system, was assessed using real-time PCR, flow cytometry and bioluminescence. The ColE7 antimicrobial system consists of the genetically modified Escherichia coli strain Nissle 1917 harbouring a conjugative plasmid (derivative of the F-plasmid) encoding the "kill" gene (ColE7 activity gene) and a chromosomally encoded "anti-kill" gene (ColE7 immunity gene). On the basis of traJ gene expression in the killer donor cells, our results showed that the efficiency of the here studied antimicrobial system against target E. coli was higher at 4 than at 24 h. Flow cytometry was used to indirectly estimate DNase activity of the antimicrobial system, as lysis of target E. coli cells in the conjugative mixture with the killer donor strain led to reduction in cell cytosol fluorescence. According to a lux assay, E. coli TG1 (pXen lux+ Apr ) with constitutive luminescence were killed already after 2 h of treatment. Target sensor E. coli C600 with DNA damage SOS-inducible luminescence showed significantly lower SOS induction 6 and 24 h following treatment with the killer donor strain. Our results thus showed that bioluminescent techniques are quick and suitable for estimation of the ColE7 bacterial conjugation-based antimicrobial system antibacterial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial antimicrobial resistance is worldwide rising and causing deaths of thousands of patients infected with multi-drug resistant bacterial strains. In addition, there is a lack of efficient alternative antimicrobial agents. The significance of our research is the use of a number of methods (real-time PCR, flow cytometry and bioluminescence-based technique) to assess the antibacterial activity of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system. Bioluminescent techniques proved to be rapid and suitable for estimation of antibacterial activity of ColE7 bacterial conjugation-based antimicrobial system and possibly other related systems.

H-NS and Lrp serve as positive modulators of traJ expression from the Escherichia coli plasmid pRK100.

Conjugative transfer of F-like plasmids is a tightly regulated process. The TraJ protein is the main positive activator of the tra operon which encodes products required for conjugative transfer of F-like plasmids. Nucleotide sequence analysis revealed potential Lrp and H-NS binding sites in the traJ regulatory region. Expression of a traJ-lacZ fusion in hns and lrp mutant strains showed that both are positive modulators of traJ expression. Competitive RT-PCR demonstrated that H-NS and Lrp exert their effect at the transcriptional level. Electrophoretic mobility-shift assays showed that H-NS and Lrp proteins bind to the traJ promoter. Conjugative transfer of pRK100 was decreased in hns but not in lrp mutant strains. Together, the results indicate H-NS and Lrp function as activators of traJ transcription.

Morphine-fluoxetine interactions in healthy volunteers: analgesia and side effects.

The authors evaluated the ability of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), to enhance the analgesic potency of morphine. Fifteen volunteers participated in this double-blind crossover study. All received combinations of morphine or saline with either fluoxetine 30 mg or placebo. The authors used individual morphine pharmacokinetics to program an infusion pump to achieve plasma morphine levels of 15, 30, and 60 ng/ml. Analgesia during morphine infusion was assessed using a model of electrical tooth stimulation. Subjective side effects, measurements of end-tidal CO2, O2 saturation, pupil size, and testing of psychomotor performance were obtained. Plasma morphine concentrations were not affected by fluoxetine. In comparison to placebo, oral fluoxetine resulted in less sedation during morphine infusion and less nausea during morphine washout. Morphine-induced pruritus, psychomotor function, and respiratory depression were unaffected by fluoxetine. Acute administration of 30 mg oral fluoxetine augmented analgesia by approximately 3% to 8% and reduced morphine-associated nausea, mood reduction, and drowsiness.

Prescription opiate abuse in chronic pain patients: clinical criteria, incidence, and predictors.

OBJECTIVES: Opiates are commonly used to treat patients with chronic nonmalignant pain. There is much controversy over the definition, incidence, and risk factors of prescription opiate abuse in chronic pain treatment. The present study, done at the Seattle VA Medical Center, was designed to create opiate abuse criteria, test inter-rater reliability of the criteria, apply the criteria to a group of chronic pain patients, and correlate the risk of opiate abuse with the results of alcohol and drug testing. DESIGN/OUTCOME MEASURES: A committee of experienced pain providers designed a five-point prescription opiate abuse checklist based on DSM-III-R parameters. The criteria were then applied to patients enrolled in the pain clinic. The reliability of the criteria were determined using two providers who were familiar with every patient in the clinic. Drug, alcohol, and psychosocial testing were correlated with the risk of opiate abuse. RESULTS: A total of 19% (76/403) of all pain clinic patients were using chronic opiates. Thirty-four percent (26/76) met one, and 27.6% (21/76) met three or more of the abuse criteria. The criteria had an inter-rater reliability of > 0.9. There were no differences between chronic opiate users (n = 76) and opiate abusers (n = 21) for a history of drug or alcohol abuse or on psychosocial testing. CONCLUSIONS: Prescription opiate abuse criteria for use in patients with chronic nonmalignant pain were designed. The criteria had good reliability and can be applied during normal clinic interactions. The percentage of chronic opiate users who become opiate abusers in pain treatment is within the range reported by others. Past opiate or alcohol abuse or psychosocial testing on clinic admission failed to predict who would become an opiate abuser. The criteria can be used to identify patients who will subsequently require more intensive treatment or intervention or can be used as an outcome to measure to test the effectiveness of treatment strategies.

Selective musculocutaneous nerve block and infraclavicular brachial plexus anesthesia. Case report.

BACKGROUND AND OBJECTIVES: Regional anesthesia of the upper extremity may be achieved by the infraclavicular approach to the brachial plexus. METHODS: Advantages of this approach include profound anesthesia of the upper extremity with minimal risk of complications. RESULTS: Isolated block of the musculocutaneous nerve may result by this approach if biceps muscle contractions are accepted as evidence of brachial plexus location by peripheral nerve stimulation. CONCLUSIONS: Stimulation of the musculocutaneous nerve in the infraclavicular region results in biceps muscle contraction. Inadequate anesthesia of the upper extremity may result due to exiting of the musculocutaneous nerve outside the axillary sheath in this region. Evidence of more distal stimulation (finger/wrist flexion) improves the likelihood of successful block of the brachial plexus by the infraclavicular route.

In vivo labelling of RBC with 99mTc for blood pool imaging using different stannous radiopharmaceuticals.

The in vivo 99mTc-RBC labelling efficiency and stability of labelling was assessed after pretinning with a high-stannous content DTPA kit (Sn DTPA) in comparison with Sn-pyrophosphate (Sn PPi) and a low-stannous DTPA kit (DTPA). The distribution in Sprague Dawley rats showed that similar fractions of administered 99mTc remained within the blood pool after pretinning with Sn DTPA and Sn PPi when equal quantities of stannous ions (15 micrograms/kg) and equal time intervals (30 min) between successive IV injections of pretinning agent and 99mTc-pertechnetate were used. Significantly lower fractions were found when DTPA (1.9 micrograms Sn2+/kg) was used for pretinning. The rate of 99mTc elution emphasises the importance of the Sn2+ concentration used, not only for labelling efficiency but also for stability of the labelling. Satisfactory intravascular activity, exceeding 80% during the first hour post-injection, was demonstrated in three volunteers after 99mTc injection, when Sn DTPA was used for pretinning. Left ventricular ejection fractions (LVEF) measured by equilibrium radionuclide angiography after pretinning with Sn DTPA in 24 patients correlated well (r = 0.98) with those obtained by contrast angiographies over a broad spectrum of values (0.14-0.72). Four repeated LVEF measurements at 45-min intervals in six additional patients at rest showed excellent reproducibility in each patient: maximum variation was less than 6%.

Comparative accumulation of 99mTc and 131I in thyroid nodules: case report.

Imaging was performed with both pertechnetate and 131I in 58 patients with thyroid nodules. Pertechnetate concentrated in all 12 follicular carcinomas, in two out of seven papillary carcinomas, and in some benign nodules that did not accumulate 131I in the 24-hr images.