Epidemiology of antimicrobial resistance in commensal E. coli from healthy dairy cattle on a Mediterranean pasture-based system of Australia: A cross-sectional study.

This study aimed to determine the prevalence of antimicrobial resistance (AMR) in commensal E. coli from healthy lactating cows and calves in the Mediterranean pasture-based feeding dairy system of Western Australia (WA). Fecal samples were collected from healthy adult lactating cows and healthy calves from dairy farms in WA. Presumptive commensal E. coli was isolated from these samples and confirmed using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Broth microdilution was used to assess the prevalence and the phenotypic AMR profiles of the E. coli isolates to 8 antimicrobial agents of dairy industry and human importance. The minimum inhibitory concentration (MIC) for each isolate was interpreted using the Epidemiologic Cutoff (ECOFF) and Clinical and Laboratory Standards Institute (CLSI) breakpoints. Genomic characterization provided multi-locus sequence types and AMR genes for a selection of isolates categorised as non-wild type (NWT) by ECOFF values for the combination of ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. From a total of 1,117 fecal samples (633 adult, 484 calf) collected across 26 randomly selected farms, 891 commensal E. coli isolates were recovered (541 adult, 350 calf). Commensal E. coli classified as NWT was highest for ampicillin for both adult (68.8%; 95% CI = 64.7 - 72.7) and calf feces (67.1%; 95% CI = 62.0 - 72.0). A large proportion of tetracycline NWT and trimethoprim-sulfamethoxazole NWT organisms were also identified from calf feces, being 44.0% (95% CI = 38.7 - 49.4) and 24.6% (95% CI = 20.2 - 29.4) respectively. Clinical resistance prevalence was low, being higher for calves than for adult feces (ampicillin (adult: 7.8% (95% CI = 5.7 - 10.3); calf: 30.0% (95% CI = 25.2 - 35.1), tetracycline (adult: 6.3% (95% CI = 4.4 - 8.7); calf: 40.3% (95% CI = 35.1 - 45.6), and trimethoprim-sulfamethoxazole (adult: 2.6% (95% CI = 1.4 - 4.3); calf: 22.0% (95% CI = 17.7 - 26.7)). Commensal E. coli originating from calf feces was significantly higher in NWT prevalence compared with adult feces for ciprofloxacin (P = 0.023), gentamicin (P = 0.02), tetracycline (P < 0.001), and trimethoprim-sulfamethoxazole (P < 0.001). The overall number of antimicrobials an isolate was classified as NWT toward varied among farms and was significantly higher for isolates originating from calf than adult feces (P < 0.001). The strain type and sampling source of the commensal E. coli investigated were both associated with the commonality of the resultant resistance genome. Clinical resistance and NWT classification were highest for ampicillin, tetracycline, and trimethoprim-sulfamethoxazole, all antimicrobials commonly used in the treatment of dairy cattle in Australia. Though highly variable across farms, commensal E. coli isolated from healthy dairy calf feces had significantly higher NWT and multidrug resistance (MDR) prevalence compared with feces from healthy adult lactating dairy cows. The resistant genome identified in MDR isolates, though not always consistent with the phenotype, included QnrS1 and genes encoding AmpC ß-lactamase and aminoglycoside phosphotransferase.

Low density lipopolyprotein inhibits flavivirus acquisition in Aedes aegypti.

Aedes aegypti is the primary vector of a number of human pathogens including dengue virus (DENV) and Zika virus (ZIKV). Ae. aegypti acquires these viruses during the processing of bloodmeals obtained from an infected vertebrate host. Vertebrate blood contains a number of factors that have the potential to modify virus acquisition in the mosquito. Interestingly, low density lipopolyprotein (LDL) levels are decreased during severe DENV infection. Accordingly, we hypothesized that LDL is a modifiable factor that can influence flavivirus acquisition in the mosquito. We found that LDL is endocytosed by Ae. aegypti cells in a dynamin-dependent manner. LDL is also endocytosed by midgut epithelial cells and accumulates at the luminal midgut epithelium during bloodmeal digestion. Importantly, pretreatment with LDL, but not high density lipopolyprotein (HDL), significantly inhibited both DENV and ZIKV infection in vitro, and LDL inhibited ZIKV infection in vivo. This study identifies human LDL or 'bad cholesterol' as a modifiable factor that can inhibit flavivirus acquisition in Ae. aegypti. Identification of modifiable blood factors and critical cellular interactions that mediate pathogen acquisition may lead to novel strategies to disrupt the transmission cycle of vector-borne diseases.

[des-Asp1]angiotensin II in the dog: blood levels and effect on aldosterone.

Circulating levels of [des-Asp1]angiotensin II ([des-Asp1]-AII), angiotensin II (AII), and aldosterone were measured in five conscious beagle dogs before and during iv infusion of [des-Asp1]AII at rates of 3, 6, 12, and 24 ng/kg/min. The animals were studied after 4 days on a normal sodium and potassium diet and again after a period of sodium depletion accomplished by iv furosemide (2-5 mg/kg) and 4 days of low sodium diet (2-5 mmol/day). Compared to the normal sodium diet, sodium depletion resulted in increases in the plasma levels of aldosterone from 10 +/- 2 (SE) to 66 (16-116) ng/100 ml of AII from 16 +/- 4 to 52 +/- 13 pmol/liter and of [des-Asp1]AII from 2 +/- 0.7 to 12 +/- 4 nmol/liter. Incremental infusions of [des-Asp1]AII in the sodium replete state resulted in progressive increases in the plasma levels of aldosterone in all dogs. In comparison with a previous study in which dogs were infused with AII, it was apparent that [des-Asp1]AII was equally or slightly more potent in stimulating aldosterone and had a higher metabolic clearance rate than AII. [des-Asp1]AII stimulated aldosterone in four of the five sodium-depleted dogs but no steepening of the [des-Asp1]AII/aldosterone dose-response curves was apparent. These results do not support the hypothesis that circulating [des-Asp1]AII mediates the effect of AII on aldosterone in the dog.

Angiotensin II/aldosterone dose-response curves in the dog: effect of changes in sodium balance.

The possibility that the responsiveness of plasma aldosterone concentration to angiotensin II alters with changes in sodium balance was investigated in male beagle dogs under conditions of controlled sodium and potassium intake. Angiotensin II was infused at four different rates (usually 3, 6, 12, and 24 ng/kg/min), each for 1 h, 1) after periods of normal sodium diet (32 mEq/day), 2) after moderate sodium depletion (negative cumulative sodium balance 25-58 mEq), 3) after severe sodium depletion (65-116 mEq negative cumulative sodium balance), and 4) after sodium loading (150-212 mEq positive sodium balance), daily potassium intake remaining constant (26 mEq/day) throughout. Angiotensin II/aldosterone dose-response curves after moderate sodium depletion were both elevated and steepened in comparison with those found during normal sodium intake. Severe sodium depletion was associated with even greater elevation of dose-response curves, but individual aldosterone responses to angiotensin II were irregular and unpredictable. Sodium loading significantly diminished aldosterone responsiveness to angiotensin II. Blood pressure increments during angiotensin II infusion were attenuated by sodium depletion.

H-77: a potent new renin inhibitor. In vitro and in vivo studies.

Chemical modification of the backbone at the cleavage site in the (6-13)-octapeptide of equine angiotensinogen resulted in greatly increased binding affinity and resistance to cleavage by renin. The D-His6-Tyr13 octapeptide analog containing the reduced bond -CH2-NH-instead of a peptide bond -CO-NH- at the Leu10-Leu11 linkage (H-77) was a powerful in vitro inhibitor of canine renin (IC50 = 24nM). It gave an IC50 of 1 microM against human renin and 0.6 microM against rat renin. In sodium-depleted conscious dogs, infusion of H-77 caused dose-related falls of plasma angiotensin I plasma angiotensin II concentration and mean arterial pressure; the minimum effective dose was 0.1 mg . kg-1 hr-1. Similar infusions of H-77 in chronically catheterized rats have no effect on blood pressure or plasma angiotensin II concentration. Thus, the in vitro effect of H-77 as an inhibitor of renin in dog, human, and rat plasma was paralleled by its action in the whole animal.

Atrial natriuretic peptides and renin release.

The relationship between endogenous plasma concentrations of atrial natriuretic peptide and renin was examined in resting normal subjects and patients with cardiac impairment. To test the hypothesis that atrial natriuretic peptide inhibits renin secretion, intravenous infusions of atrial natriuretic peptide were administered to normal volunteers, patients with end-stage renal failure, and conscious dogs in both sodium-replete and sodium-depleted states. Plasma atrial natriuretic peptide and renin were inversely related in normal subjects (r = -0.52, n = 140, p less than 0.001), but a weak positive association between these two variables was observed in patients with cardiac impairment (r = 0.32, n = 60, p less than 0.02). Low doses of both 26- and 28-amino-acid human atrial natriuretic peptide (2 pmol/kg/minute for two hours) given to sodium-replete normal subjects halved plasma renin compared with time-matched placebo values (19 +/- 4 and 18 +/- 3 versus 36 +/- 8 microU/ml, p less than 0.001 for both). Incremental doses of synthetic atrial natriuretic peptide suppressed plasma renin below time-matched placebo values in both sodium-replete (maximal suppression 1.2 +/- 0.4 versus 8.6 +/- 1.4 microU/ml, p less than 0.001) and sodium-depleted (maximal suppression 18.9 +/- 4.9 versus 51 +/- 13 microU/ml, p less than 0.05) dogs. This effect was initially apparent at low doses of atrial natriuretic peptide (1 pmol/kg/minute), and renin suppression was maximal, in both states, with lesser doses of atrial natriuretic peptide than those at which maximal natriuresis was observed. Atrial natriuretic peptide administered to patients with end-stage renal failure (10 pmol/kg/minute for one hour) caused no change in plasma renin. These data confirm that atrial natriuretic peptide inhibits renin secretion in a dose-related manner and suggest that this action of the peptide is modified by both the baseline sodium status and renal function of the recipient.

Similarity between active and trypsin-activated inactive renin in dog plasma by means of renin inhibition: the dog as an animal model for studies of inactive renin.

A method for trypsin-activation of dog plasma inactive renin is described. Liquid phase trypsin (final concentration 6.7 mg/ml) was used and the reaction was stopped after 2 min at 4 degrees C by soybean trypsin inhibitor (13 mg/ml). Renin was measured as angiotensin I (Ang I) generation in trypsin-treated and untreated plasma using the antibody-trapping method, in the presence of excess ox renin substrate. The renin-like activity after trypsin was indeed due to renin, since Ang I generation in dog plasma before and after trypsin treatment was completely inhibited by H-77 at 10(-6) mol/l, and the two IC50 values were very similar (2.7 +/- 0.7 and 2.9 +/- 0.7 at 10(-8) mol/l, respectively). Dog plasma inactive renin was effectively separated from active renin by chromatography on Affigel Blue. Like human prorenin, dog plasma inactive renin rose in response to sodium depletion (furosemide 5 mg/kg, i.v.) followed by a low-salt diet (1 mmol Na+/day) for 4 days, (from 29.6 +/- 8 to 162 +/- 22 microU/ml; P less than 0.01, n = 10). Active renin also increased as expected. Intravenous captopril (6 mg/kg per h), for 3 h, led to a sharp increase in dog plasma active renin (from 53 +/- 8 to 360 +/- 60 microU/ml; P less than 0.01, n = 6), whereas inactive renin remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

A transition-state analogue inhibitor of human renin (H.261): test in vitro and a comparison with captopril in the anaesthetized baboon.

H.261, a new transition state inhibitor of human renin with an IC50 of 6.9 X 10(-10) M, was given by intravenous infusion to six anaesthetized baboons. The inhibitor was infused first at 0.1 mumol/kg/h for 15 min, then at 1.0 mumol/kg/h for a further 15 min. After a recovery period of 2 h in which the animals received 5% dextrose, they were infused with captopril, 25 mumol/kg/h for 15 min. At both rates of infusion H.261 markedly and significantly reduced the enzymatic action of renin in plasma, the blood concentration of angiotensin I, the plasma concentration of angiotensin II and mean arterial pressure. All changes reverted towards or to control values in the subsequent control period. Captopril also lowered plasma angiotensin II concentration and mean arterial pressure markedly and significantly but, as expected for an inhibitor of the angiotensin I-converting enzyme, plasma active renin concentration and blood angiotensin I concentration increased. The changes of angiotensin II and arterial pressure were similar with captopril and H.261.

New inhibitors of human renin tested in vitro and in vivo in the anaesthetized baboon.

A new inhibitor of human renin (H. 189) is described. It is a decapeptide analogue of human renin substrate with the amino acid, statine, substituted for leucine in the scissile bond. Its inhibitory potency as shown by IC50 is 1.0 X 10(-8) M with human plasma renin and 1.5 X 10(-8) M with baboon plasma renin. It is less effective with dog and rat renin, but its inhibitory potency with human renin is similar to that of another inhibitor of ours (H. 142) having a reduced isostere in the scissile bond. H. 189 has some inhibitory effect on cathepsin D (IC50 6.5 X 10(-5) M) but H. 142 has no discernible effect. Pepstatin, on the other hand, was highly effective against cathepsin D (IC50 1.2 X 10(-8) M). H. 142 and H. 189 were infused intravenously at 10 mg/kg/h in four anaesthetized salt-deplete baboons (Papio hamadryas). The activity of renin in plasma decreased markedly as did the circulating concentration of its products, angiotensin I and angiotensin II.

Effects of prolonged and brief infusions of noradrenaline on arterial pressure and on the plasma concentrations of active renin, angiotensin II, aldosterone and potassium.

Conscious male beagle dogs were given constant intravenous infusions of noradrenaline for 14 days, four receiving 125 ng/kg/min and four 250 ng/kg/min. Before, during and after these infusions dose-response studies were done in which additional noradrenaline was infused at 500, 1000 and 2000 ng/kg/min, each rate for 1 h. Blood samples were taken before and during infusions for measurement of haematocrit and plasma concentrations of noradrenaline, active renin, angiotensin II, aldosterone, sodium and potassium. Fourteen-day infusion of noradrenaline at 125 ng/kg/min did not raise blood pressure significantly though infusion at 250 ng/kg/min did, but for the first week of infusion only. Heart rate decreased significantly at both rates. Arterial pressure fell markedly and significantly on stopping infusion. Mean plasma concentrations of renin, angiotensin II and aldosterone tended to be lower during prolonged infusion of noradrenaline, but only the fall of renin during the second week was significant in one group of dogs. Noradrenaline at higher rates significantly raised blood pressure and increased plasma concentrations of renin and angiotensin II. Plasma aldosterone concentration did not rise significantly, perhaps because plasma potassium concentration decreased; in support of this theory changes of plasma aldosterone correlated with changes of plasma potassium but not with changes of angiotensin II. The rise in arterial pressure during dose-response studies was related to the increase of plasma noradrenaline. Prolonged infusion of noradrenaline did not alter the dose-response relation between plasma noradrenaline concentration and arterial pressure.

Effect of infused captopril on blood pressure and the renin-angiotensin-aldosterone system in normal dogs subjected to varying sodium balance.

Infusion of captopril at 20, 200, 2,000 and 6,000 micrograms/kg/hour into sodium-depleted conscious dogs produced a rapid, dose-dependent decrease in blood pressure and plasma angiotensin II and III, maximal suppression being achieved at 200 micrograms/kg/hour (97 +/- 14 to 65 +/- 8 [standard deviation] mm Hg, 38 +/- 10.6 to 3.2 +/- 1.5 pmol/liter and 7.0 +/- 4.8 to 1 +/- 0.5 pmol/liter, respectively). Angiotensin I concentration increased with each infusion rate to a maximal 16-fold increase at 6,000 micrograms/kg/hour (26 to 416 pmol/liter). For all infusion rates the percentage decrease in blood pressure correlated with the percentage decrease in plasma angiotensin II (r = 0.65, p less than 0.001). Infusion of captopril at 6,000 micrograms/kg/hour into sodium-loaded dogs also produced a decrease in both blood pressure (117 +/- 9 to 96.6 +/- 11 mm Hg) and plasma angiotension II (11.0 +/- 3 to 1.6 +/- 1.3 pmol/liter). Plasma aldosterone concentrations decreased whereas both blood angiotensin I and renin concentration increased. In another experiment angiotensin II was infused at 2, 6, 18 and 54 ng/kg/min into sodium-depleted dogs firstly without modification and secondly combined with captopril (6,000 micrograms/kg/hour) given for 1 hour before the angiotensin dose-response study and continued throughout. Angiotensin II infusion raised mean arterial pressure and plasma angiotensin II in each animal. However, the angiotensin II blood pressure dose-response curve was shifted downwards and to the right in the captopril-treated animals. These results suggest that arterial pressure and aldosterone secretion in normal dogs are partly dependent on the renin-angiotensin system but that not all of the acute decrease in blood pressure produced by captopril can be explained by the suppression of the acute vasoconstrictor effect of circulating angiotensin II.

A microassay for active and total renin concentration in human plasma based on antibody trapping.

We have developed and validated a new enzyme-kinetic method for measurement of renin concentration (PRC) in human plasma, based on radioimmunoassay of angiotensin I generated during incubation of plasma and excess sheep or ox renin substrate. Angiotensin I breakdown during incubation is prevented by the presence of anti-angiotensin I serum. The assya does not require prior extraction of renin, is technically simple, and is sufficiently sensitive to measure subnormal renin levels. With minor modifications both "active and "total" renin may be measured. Assay results have been calibrated with the International Standard Renin. PRC measured by this technique correlates significantly with angiotensin I and II, plasma renin activity, and with the PRC method previously used by us.

Plasma angiotensin II, renin, renin-substrate and aldosterone concentrations in acute renal failure in man.

Plasma concentrations of angiotensin II, renin, renin-substrate and aldosterone were measured in cases of acute renal failure. Angiotensin II, and renin levels were abnormally high on at least one occasion in nearly all patients. Mean angiotensin II and renin levels were highest in the first ten days of the disease. There was a highly significant positive correlation between concurrent estimations of renin and angiotensin II. Renin-substrate was also frequently elevated, but the correlations with renin and angiotensin II were not statistically significant. Despite the frequently marked elevation of plasma angiotensin II, only 2 of 17 measurements of plasma aldosterone were abnormally high. There was no significant relationship between aldosterone and plasma concentrations of angiotensin II, renin, sodium or potassium. The data are discussed in relation to current hypotheses implicating renin and angiotensin in the pathogenesis of acute circulatory renal failure.

Occupational lead exposure and renin release.

Hypertension may result from chronic lead exposure. Lead poisoning arising from "moonshine whiskey" drinking has been associated with a rise in plasma renin activity. In the present study, plasma renin concentration following intravenous administration of frusemide was measured in eleven subjects with moderate or severe lead poisoning of industrial origin. The results were compared with those obtained for seven normal, control subjects. There was no significant difference in response obtained in the two groups. Industrial lead poisoning does not appear to affect renin release. The combined insult of lead and alcohol may explain the findings in the previous study.

Peptide inhibitors of renin.

Analogues of renin substrate that incorporated a non-cleavable isostere at the scissile bond are powerful inhibitors of renin both in vitro, and in animals and man.

Effects of the renin inhibitor H77 on angiotensin II, arterial pressure and cardiac function in conscious dogs: comparison with captopril.

The renin inhibitor H77 and the angiotensin I converting enzyme (ACE) inhibitor captopril were compared in separate experiments with infusion of 5% dextrose as a control for the effects on plasma angiotensin II (Ang II) concentration, arterial pressure and cardiac function, measured by Swan-Ganz catheter, in conscious dogs. The effects of a high dose of H77 (10 mg/kg per h) were similar to those of high-dose captopril (6 mg/kg per h). Both reduced plasma Ang II concentration, systemic vascular resistance and arterial pressure; both increased the heart rate; both increased cardiac output but the change was significant only with captopril; neither affected stroke volume, pulmonary artery pressure or pulmonary vascular resistance; both reduced left and right atrial pressures. The similar pattern of effects for the two inhibitors suggests that the mechanism by which they act is the same--reduction in Ang II--and that the cardiovascular effects of H77 are not a specific action of the peptide that is unrelated to the reduction in plasma Ang II concentrations.