The Role of AcrAB-TolC Efflux Pumps on Quinolone Resistance of E. coli ST131.

Escherichia coli ST131 is a cause for global concern because of its high multidrug resistance and several virulence factors. In this study, the contribution of acrAB-TolC efflux system of E. coli ST131 to fluoroquinolone resistance was evaluated. A total of nonrepetitive 111 ciprofloxacin-resistant E. coli isolates were included in the study. Multilocus sequence typing was used for genotyping. Expressions of acrA, acrB, and TolC efflux pump genes were measured by RT-PCR. Mutations in marA, gyrA, parC, and aac(6')-lb-cr positivity were studied by Sanger sequencing. Sixty-four (57.7%) of the isolates were classified as ST131, and 52 (81.3%) of the ST131 isolates belonged to H30-Rx subclone. In ST131, CTX-M 15 positivity (73%) and aac(6')-lb-cr carriage (75%) were significantly higher than those in non-ST131 (12.8% and 51%, respectively) (P < 0.05). The ampicillin-sulbactam (83%) resistance was higher, and gentamicin resistance (20%) was lower in ST131 than that in non-ST131 (64% and 55%, respectively) (P = 0.001 and P = 0.0002). Numbers of the isolates with MDR or XDR profiles did not differ in both groups. Multiple in-dels (up to 16) were recorded in all quinolone-resistant isolates. However, marA gene was more overexpressed in ST131 compared to that in non-ST131 (median 5.98 vs. 3.99; P = 0.0007). Belonging to H30-Rx subclone, isolation site, ciprofloxacin MIC values did not correlate with efflux pump expressions. In conclusion, the marA regulatory gene of AcrAB-TolC efflux pump system has a significant impact on quinolone resistance and progression to MDR profile in ST131 clone. Efflux pump inhibitors might be alternative drugs for the treatment of infections caused by E. coli ST131 if used synergistically in combination with antibiotics.

Uric acid stimulates proliferative pathways in vascular smooth muscle cells through the activation of p38 MAPK, p44/42 MAPK and PDGFRß.

Hyperuricemia and angiotensin II (Ang II) may have a pathogenetic role in the development of hypertension and atherosclerosis as well as cardiovascular disease (CVD) and its prognosis. The purpose of this study was to investigate whether uric acid can induce proliferative pathways of vascular smooth muscle cell (VSMC) that are thought to be responsible for the development of CVD. The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and platelet-derived growth factor receptor ß (PDGFRß) was measured by Elisa and Western blot techniques to determine the activation of proliferative pathways in primary cultured VSMCs from rat aorta. Results demonstrated that uric acid can stimulate p38 MAPK, p44/42 MAPK and PDGFRß phosphorylation in a time- and concentration-dependent manner. Furthermore, treatment of VSMCs with the angiotensin II type I receptor (AT1R) inhibitor losartan suppressed p38 MAPK and p44/42 MAPK induction by uric acid. The stimulatory effect of uric acid on p38 MAPK was higher compared to that of Ang II. The results of this study show for the first time that uric acid-induced PDGFRß phosphorylation plays a crucial role in the development of CVDs and that elevated uric acid levels could be a potential therapeutical target in CVD patients.

Healthcare-associated Gram-negative bloodstream infections: antibiotic resistance and predictors of mortality.

This article describes the prevalence of antibiotic resistance and predictors of mortality for healthcare-associated (HA) Gram-negative bloodstream infections (GN-BSI). In total, 831 cases of HA GN-BSI from 17 intensive care units in different centres in Turkey were included; the all-cause mortality rate was 44%. Carbapenem resistance in Klebsiella pneumoniae was 38%, and the colistin resistance rate was 6%. Multi-variate analysis showed that age >70 years [odds ratio (OR) 2, 95% confidence interval (CI) 1.22-3.51], central venous catheter use (OR 2.1, 95% CI 1.09-4.07), ventilator-associated pneumonia (OR 1.9, 95% CI 1.1-3.16), carbapenem resistance (OR 1.8, 95% CI 1.11-2.95) and APACHE II score (OR 1.1, 95% CI 1.07-1.13) were significantly associated with mortality.

Antimicrobial susceptibilities of uropathogen Escherichia coli in renal transplant recipients: dramatic increase in ciprofloxacin resistance.

The urinary tract is the most common site of bacterial infections in renal transplant recipients. The management of urinary tract infections (UTI) in renal transplant recipients is becoming more difficult because of drug-resistant bacteria. The antimicrobial susceptibilities of uropathogen bacteria isolated from 398 patients who underwent renal transplantation between 2007 and 2011 were obtained from medical records. At least 1 UTI episode was diagnosed in 172 (43.2%) patients. Among the 703 bacteria isolated from these patients, Exherichia coli the most common pathogen, was isolated from 407/703 episodes (57.8%). Ciprofloxacin, co-trimoxazole, ceftriaxone, and gentamicin resistance rates were 59.4%, 85.7%, 40.7%, and 36.6%, respectively. Ninty six of 407 E. coli isolates (23.5%) were ESBL positive. Analysis of resistance rates in our center demonstrated ciprofloxacin resistance rate in uropathogenic E. coli to have increased gradually from 30.4% in 2003, 41.3% in 2007, and 59.4% in 2012. Instutional data regarding the etiologic agents and antimicrobial susceptibility results are important for proper management of patients with UTI.

Plasma levels of nitrites, PGF1alpha, and nitrotyrosine in LPS-treated rats: functional and histochemical implications in aorta.

We investigated the effects of lipopolysaccharide (LPS) administration on plasma nitrite, nitrotyrosine and 6-keto prostaglandin F1alpha, (PGF1alpha) levels and the related resultant changes in function and histochemistry of aorta in rats. Plasma nitrite and PGF1alpha nitrotyrosine levels were analysed after 5 mg/kg intravenous LPS was administered to rats compared with those in non-treated rats. The distribution of nitrotyrosine in the aorta was studied immunohistochemically. The contractile responses of aortic rings to phenylephrine (PE) from both the LPS-treated and control rats were studied in the organ baths. There were increases in plasma nitrite, PGF1alpha, and nitrotyrosine concentrations of LPS-treated rats compared to non-treated rats. Immunoreactivity of nitrotyrosine residues were detected in the endothelial and smooth muscle cells in LPS-treated but not in control rat aorta. The contractile responses to PE of the LPS-treated rat aortic rings were significantly reduced as compared with those of control rat's. Incubation of the aortic rings from LPS-treated rats with cyclooxygenase inhibitor indomethacine or with a combination of indomethacine and nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) increased the contractile responses to the levels observed in control rats suggesting that both prostanoids and particularly nitric oxide (NO) are involved in the reduced contractile responses in LPS-treated rats. These results supported the view that LPS might cause an increment in both NO and PGI2 levels. This increase in the NO and PGI2 levels may be responsible from the reduction in responses of aorta to contractile agents in LPS-treated rats. Increased peroxynitrite formation in LPS-treated rats may lead to nitration of the tyrosil residues of the proteins in the aorta.

Plasma levels of nitrites, PGF1 alpha and nitrotyrosine in LPS-treated rats: functional and histochemical implications in aorta

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We investigated the effects of lipopolysaccharide (LPS) administration on plasmanitrite, nitrotyrosine and 6-keto prostaglandin F1 alpha (PGF1 alpha) levels and the relatedresultant changes in function and histochemistry of aorta in rats. Plasma nitrite and PGF1 alpha, nitrotyrosine levels were analysed after 5 mg/kg intravenous LPS was administered to rats compared with those in non-treated rats. The distribution of nitrotyrosinein the aorta was studied immunohistochemically. The contractile responses of aorticrings to phenylephrine (PE) from both the LPS-treated and control rats were studiedin the organ baths. There were increases in plasma nitrite, PGF1á, and nitrotyrosineconcentrations of LPS-treated rats compared to non-treated rats. Immunoreactivityof nitrotyrosine residues were detected in the endothelial and smooth muscle cellsin LPS-treated but not in control rat aorta. The contractile responses to PE of the LPStreatedrat aortic rings were significantly reduced as compared with those of controlrat’s. Incubation of the aortic rings from LPS-treated rats with cyclooxygenaseinhibitor indomethacine or with a combination of indomethacine and nitric oxide synthaseinhibitor Nù-nitro-L-arginine methyl ester (L-NAME) increased the contractileresponses to the levels observed in control rats suggesting that both prostanoids andparticularly nitric oxide (NO) are involved in the reduced contractile responses inLPS-treated rats. These results supported the view that LPS might cause an incrementin both NO and PGI2 levels. This increase in the NO and PGI2 levels may be responsiblefrom the reduction in responses of aorta to contractile agents in LPS-treated rats.Increased peroxynitrite formation in LPS-treated rats may lead to nitration of thetyrosil residues of the proteins in the aorta

Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats.

Oxidant stress is one of the factors proposed to be responsible for damaged erythrocytes observed during and after exercise. The impact of exertional oxidant stress after acute exhaustive treadmill running on erythrocyte damage was investigated in sedentary (Sed) and exercise-trained (ET) rats treated with or without antioxidant vitamins C and E. Exhaustive exercise led to statistically significant increments in the levels of thiobarbituric acid-reactive substance (TBARS) and H2O2-induced TBARS in Sed rats and resulted in functional and structural alterations in erythrocytes (plasma hemoglobin concentrations, methemoglobin levels, and rise in osmotic fragility of erythrocytes with decrease in erythrocyte deformability). Administration of antioxidant vitamin for 1 mo before exhaustive exercises prevented lipid peroxidation (TBARS, H2O2-induced TBARS) in Sed rats without any functional or structural alterations in erythrocytes. Parameters indicating erythrocyte lipid peroxidation and deterioration after exhaustive exercise in rats trained regularly with treadmill running for 1 mo were not different from those in Sed controls. Erythrocyte lipid peroxidation (TBARS) increased in exhausted-ET rats compared with ET controls; however, the plasma hemoglobin, methemoglobin levels, and erythrocyte osmotic fragility and deformability did not differ. Exhaustive exercise-induced lipid peroxidation in ET rats on antioxidant vitamin treatment was prevented, whereas functional and structural parameters of erythrocytes were not different from those of the ET controls. We conclude that exertional oxidant stress contributed to erythrocyte deterioration due to exercise in Sed but not in ET rats.

Alterations in the erythrocyte antioxidant system of blood stored in blood bags.

In the present study, we measured the concentrations of reduced glutathione (GSH) and malonyldialdehyde (MDA) and the activities of glutathione peroxidase (GSH-Px), glutathione S-transferase (GSH-S-T), superoxide dismutase (SOD), catalase (CAT) and glucose-6-phosphate dehydrogenase (G-6-PD) in erythrocytes obtained freshly from adult male donors which was preserved with CPDA-1 anticoagulant (citrate,phosphate, dextrose, adenine) on different days of storage. At the end of the study, storage-associated alterations in antioxidant activities were noted and discussed. GSH, GSH-Px, GSH-S-T, SOD, CAT and G-6-PD activities decreased, but erythrocyte MDA levels, as anindex of lipid peroxidation, increased during the storage period. According to our results, glutathione-dependent antioxidant systems in erythrocytes might be depleted during long storage in blood bags.

Effect of intraportal verapamil infusion on hepatic ischemia-reperfusion injury.

Removal of free oxygen radicals, generated during reperfusion of an ischemic organ by scavengers protects the tissue from reperfusion injury. The calcium channel blocker verapamil is an effective cytoprotective agent, preventing against reperfusion injury. The effects of verapamil were investigated previously using hepatic, renal or cardiac ischemia-reperfusion injury models. We investigated the effects of intravenous and intraportal administration of verapamil in prevention from the injury caused by free oxygen radicals generated during hepatic ischemia-reperfusion in rats. Thirty six male Sprague-Dawley rats after laparotomy were subjected to hepatic ischemia for 30 and 45 min followed by 60 min of reperfusion. Two minutes before ischemia the rats were pretreated by intravenous or intraportal administration of verapamil. The levels of glutathione and thiobarbituric acid reacting substances (TBARs) referred to as malonyldialdehyde (MDA) and the serum levels of transaminases were measured in liver tissue 1 and 24 h after the onset of reperfusion. Statistical analysis of the data by Student's t-test showed statistically significant differences between the group pretreated intraportally with verapamil and the other groups. Verapamil given intraportally exerted more beneficial effect. Therefore, we conclude that intraportal verapamil administration reduces the ischemia-reperfusion injury caused by free oxygen radicals.

Red blood cell rheological alterations in hypertension induced by chronic inhibition of nitric oxide synthesis in rats.

Nitric oxide (NO) plays a major role in vascular regulation. Modulation of NO synthesis is known to influence blood pressure. Inhibition of NO synthesis by NG-nitro-L-arginine methyl ester (L-NAME; 72 mg/kg/day, p.o., 21 days) resulted in 60% increase in blood pressure in rats. Red blood cell (RBC) transit time measured by the cell transit analyzer increased significantly in the L-NAME treated group, in comparison to normotensive rats. RBC aggregation measured in autologous plasma, by a photometric rheoscope also increased significantly in the hypertensive rats. RBC cytosolic free calcium concentration was also significantly higher in the hypertensive animals. Incubation of RBC from hypertensive and control animals with NO donor, sodium nitroprusside (SNP; 10-1000 microM) for 60 minutes resulted in a dose-dependent decrease in RBC aggregation, however aggregation index was significantly higher in hypertensive group at each SNP concentration. Incubation with SNP had no effect on RBC deformability in the control group, while a slight decrease in RBC transit time was observed only at 10 microM SNP in the hypertensive group. These results imply that NO may play a role in the regulation of rheological properties of RBC and the alterations in these properties may at least in part be involved in the development of L-NAME induced hypertension.

The antioxidant effect of free bilirubin on cumene-hydroperoxide treated human leukocytes.

To examine the antioxidant effect of bilirubin (BR) on leukocyte, we treated leukocytes obtained from healthy subjects with an oxidant and various concentrations of BR. High concentrations of BR decreased thiobarbituric acid reactive substances (TBARS) and catalase activities, increased superoxide dismutase (SOD) activity, but had no effect on glutathione (GSH) concentration. Our results showed that under physiological conditions, BR has an antioxidant effect only in high concentrations.

Prophylactic ranitidine in experimental fetal distress: acute phase effects on the fetal stomach.

Fetal distress (FD) adversely affects fetal gastric physiology and histology, increasing gastric acid secretion and disturbing gastric protective mechanism. Considering these findings, an experimental study was planned to test whether ranitidine prevents FD-related gastric physiological and histological changes during late gestational period. In this study, a rabbit model of FD was created by way of intermittent maternal aortic occlusion. In group 1 (SC), saline treated animals underwent control operation. In group 2 (SD), FD was created in saline treated animals. In group 3 (RC), ranitidine treated animals underwent control operation. In group 4 (RD), FD was created in ranitidine treated animals. Blood lactic acid levels of the fetuses were 2.3 +/- 1.0 mg/L in SC group and 4.7 +/- 1.8 mg/L in group SD (p < 0.01); 2.5 +/- 0.9 mg/L in group RC and 6.7 +/- 2.5 mg/L in group RD (p < 0.01). Fetal gastric acid secretion was 5.94 +/- 2.13 microEq/h in group SC and 8.26 +/- 2.24 microEq/h in group SD (p < 0.05); 6.63 +/- 2.3 microEq/h in group RC and 6.04 +/- 2.43 microEq/h in group RD (p < 0.05). Fetal gastric PGE2 level was 16.4 +/- 2.65 microg/g-wet weight in group SC and 7.62 +/- 1.86 microg/g-wet weight in group SD (p < 0.01); 15.6 +/- 2.61 microg/g-wet weight in group RC and 8.44 +/- 1.44 microg/g-wet weight in group RD (p < 0.01). In addition, histopathological examination showed normal gastric structure in groups SC and RC, but there were mild erosive and hemorrhagic changes in groups SD and RD. Because prophylactic ranitidine significantly decreased gastric acid secretion, but did not prevent harmful histopathologic effects in FD, it is suggested that gastric damage cannot be avoided by decreasing gastric acid secretion alone. However PGE2 analogs with or without H2 receptor blockers may have a potential role to prevent FD-related gastric damage.

Peroxynitrite inactivates prostacyclin synthase by heme-thiolate-catalyzed tyrosine nitration.

Previous work has shown a sensitive inhibition of prostacyclin synthase activity by peroxynitrite as well as by superoxide in the presence of NO donors. Neither superoxide nor NO alone nor decomposed peroxynitrite is effective. The inhibition of activity was paralleled by a nitration of a tyrosine residue and both could be prevented by a stable substrate analog. The same IC50 value for peroxynitrite was also found for the cellular prostacyclin activity in endothelial and kidney mesangial cells, indicating that the antioxidant potential of the cell cannot prevent the inactivation. Aortic tissue shows a co-localization of prostacyclin synthase and nitrotyrosine staining after treatment of the tissue with 1 microM peroxynitrite. It can be speculated that this pathway of enzyme nitration is of pathophysiological significance.

Deformability and oxidant stress in red blood cells under the influence of halothane and isoflurane anesthesia.

1. The effects of halothane and isoflurane anesthesia on red blood cell (RBC) deformability, lipid peroxidation and antioxidant enzymes were tested in rabbits. 2. RBC transit time was significantly increased to 2.12 +/- 0.07 msec after 1-hr halothane anesthesia preceded by 6 mg/kg pentobarbital injections from 1.98 +/- 0.07 msec preanesthesia value (p < 0.05). Thiobarbituric acid-reactive substances also were increased significantly, being 23.35 +/- 2.75 nmol/gHb and 33.11 +/- 5.34 nmol/gHb before and after anesthesia, respectively (p < 0.05). 3. Under halothane anesthesia without prior pentobarbital injection or under isoflurane anesthesia with or without pentobarbital injection, no significant alterations were observed in these parameters. 4. RBC superoxide dismutase activity was decreased in the group anesthetized with the pentobarbital-halothane combination. The impaired RBC deformability and increased oxidant damage might be related to the free radical formation during the metabolism of halothane. Pentobarbital can potentiate this effect either by inducing cytochrome P-450 or by altering antioxidant defense. 5. Alterations in RBC mechanical properties may contribute to the tissue perfusion problems that develop after surgery under general anesthesia.

Inhibition of human erythrocyte (Na(+)-K+)ATPase by organic hydroperoxides and protection by ascorbic acid and butylated hydroxytoluene.

1. The in vitro effects of cumene hydroperoxide and t-butyl hydroperoxide on intact human erythrocyte membrane (Na(+)-K+)ATPase activities have been studied. 2. (Na(+)-K+)ATPase activities on erythrocyte membranes decreased in agreement with the results of chemiluminescence experiments. 3. Our results demonstrated that the organic hydroperoxides inhibit the activity of (Na(+)-K+) ATPase enzyme and that the antioxidants used prevent this inhibition.

The effect of bilirubin on lipid peroxidation and antioxidant enzymes in cumene hydroperoxide-treated erythrocytes.

Recently, it has been suggested that bilirubin may act as a potent biological chain-breaking antioxidant. To observe the effects of free bilirubin on antioxidant reactions in cumene hydroperoxide-treated erythrocytes (15 g hemoglobin/dl), we added bilirubin at four different concentrations (0.5, 1, 5, and 10 mg/dl). We measured the thiobarbituric acid-reactive substance and reduced glutathione levels, and some antioxidant enzyme activities, namely superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase. Thiobarbituric acid-reactive substance and chemiluminescent signals decreased during the incubation. Superoxide dismutase activities also decreased but not as much as in the control group. Glucose-6-phosphate dehydrogenase activities and reduced glutathione levels increased, but catalase activities remained the same as the control group. Our results suggest that bilirubin--in the concentrations we have used--partially prevented the oxidant effects of cumene hydroperoxide.

Acute physiopathological and histopathological effects of fetal distress on the fetal stomach: an experimental study.

Although effects of stress on the stomach have been extensively investigated in children and adults, our knowledge about effects of fetal distress (FD) on the fetal stomach is quite limited. Therefore, an experimental study was planned to evaluate the effects of FD on fetal gastric physiology and histology. In this study, a model of FD was created by way of intermittent maternal aortic occlusion in pregnant rabbits. In total, 21 fetuses of 6 pregnant rabbits were available for surgical and laboratory procedures. Laboratory examinations showed that (1) fetal gastric acid secretion was 4.24 +/- 2.68 muEq/h in the control group and 18.08 +/- 6.34 muEq/h in the distress group (p < 0.01) and (2) fetal gastric PGE2 level was 16.59 +/- 6.15 mg/g wet weight in the control group and 9.86 +/- 3.46 mg/g wet weight in the distress group (p < 0.05). Histopathologically, there were mild hemorrhagic and errosive changes in the distressed fetuses, but not in control fetuses. These findings support that FD adversely affects fetal gastric physiology through two mechanisms consisting of increased gastric acid secretion and decreased fetal gastric protection in rabbits. Consequently, gastric injury should be noted as a potential problem among hypoxia-associated abnormalities encountered in the distressed fetus.

Increased resistance to oxidative stress in normal and glucose-6-phosphate dehydrogenase-deficient hemolysates in the presence of enzyme substrates.

Erythrocytes and hemolysates from 10 normal and 10 glucose-6-phosphate dehydrogenase-deficient individuals were incubated with cumene hydroperoxide, and free radical-induced lipid peroxidation was monitored by chemiluminescence. Chemiluminescence intensities in erythrocytes of normal and deficient subjects were similar in the presence or absence of glucose-6-phosphate dehydrogenase substrates. Hemolysates of normal and deficient subjects also showed similar chemiluminescence in the absence of substrates. However, with the addition of substrates to the incubation medium, deficient hemolysates reached maximum chemiluminescence intensity within a shorter period, and maximum values were higher than in normal hemolysates. We believe this offers a new means of detection of glucose-6-phosphate dehydrogenase-deficient patients.

The role of antioxidants in the prevention of t-butyl hydroperoxide-induced chemiluminescence.

An experimental system which assesses the antioxidant potential of ascorbic acid, glutathione, uric acid, and taurine was developed. The system comprised hemoglobin, luminol, t-butyl hydroperoxide, and different concentrations of antioxidants in TRIS-HCl buffer (pH 7.4). Control assays were performed by excluding antioxidants. Chemiluminescence was detected using a liquid scintillation counter in single photon mode. All antioxidants, when applied in the appropriate concentrations, decreased the maximum chemiluminescence values. The minimum concentrations which decreased the chemiluminescence values were defined for each of the antioxidants.