Search

Background: The appearance of MDR strains and the development of biofilms make Pseudomonas aeruginosa infections a therapeutic challenge. To overcome this scenario, bacteriocins have been proposed as a potential adjuvant or alternative to antibiotic treatment., Objectives: To study the activity of R-pyocins on biofilms and in a murine model of pneumonia using a high-risk clone of P. aeruginosa., Methods: The activity of R-pyocins on P. aeruginosa biofilms was tested on bacteria attached to a silicone surface, before and after biofilm formation. The effectiveness of R1-pyocin was studied in a murine model of pneumonia using ST175, a high-risk clone of P. aeruginosa., Results: R-pyocins attacked adherent bacteria, preventing biofilm formation, and penetrated into the biofilm, killing P. aeruginosa within it, resulting in a dramatic reduction in bacterial load. R1-pyocin was active in a murine model of P. aeruginosa lung infection, administered before infection as a preventive treatment, and in acute pneumonia, with efficiency higher than standard colistin treatment. In addition, this work is the first to describe histopathological lung changes after administration of R-pyocins, contributing to the resolution of P. aeruginosa pneumonia in a murine model., Conclusions: This work highlights the potential use of the R-pyocins as therapeutic agents, alone or as adjuvants, due to its effectiveness on biofilms and in a murine model of pneumonia using ST175, a high-risk clone of P. aeruginosa. It may thus be feasible to consider R-pyocins as a possible therapeutic alternative in XDR infections, where treatment alternatives are limited., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: Escherichia coli is characterized by three resistance patterns to β-lactams/β-lactamase inhibitors (BLs/BLIs): (i) resistance to ampicillin/sulbactam and susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (RSS); (ii) resistance to ampicillin/sulbactam and amoxicillin/clavulanic acid, and susceptibility to piperacillin/tazobactam (RRS); and (iii) resistance to ampicillin/sulbactam, amoxicillin/clavulanic acid and piperacillin/tazobactam (RRR). These resistance patterns are acquired consecutively, indicating a potential risk of developing resistance to piperacillin/tazobactam, but the precise mechanism of this process is not completely understood., Methods: Clinical isolates incrementally pressured by piperacillin/tazobactam selection in vitro and in vivo were used. We determined the MIC of piperacillin/tazobactam in the presence and absence of piperacillin/tazobactam pressure. We deciphered the role of the blaTEM genes in the new concept of extended-spectrum resistance to BLs/BLIs (ESRI) using genomic analysis. The activity of β-lactamase was quantified in these isolates., Results: We show that piperacillin/tazobactam resistance is induced in E. coli carrying blaTEM genes. This resistance is due to the increase in copy numbers and transcription levels of the blaTEM gene, thus increasing β-lactamase activity and consequently increasing piperacillin/tazobactam MICs. Genome sequencing of two blaTEM-carrying representative isolates showed that piperacillin/tazobactam treatment produced two types of duplications of blaTEM (8 and 60 copies, respectively). In the clinical setting, piperacillin/tazobactam treatment of patients infected by E. coli carrying blaTEM is associated with a risk of therapeutic failure., Conclusions: This study describes for the first time the ESRI in E. coli. This new concept is very important in the understanding of the mechanism involved in the acquisition of resistance to BLs/BLIs., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

An expanded library of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been constructed using the spectra generated from 42 clinical isolates and 11 reference strains, including 23 different species from 8 sections (16 cryptic plus 7 noncryptic species). Out of a total of 379 strains of Aspergillus isolated from clinical samples, 179 strains were selected to be identified by sequencing of beta-tubulin or calmodulin genes. Protein spectra of 53 strains, cultured in liquid medium, were used to construct an in-house reference database in the MALDI-TOF MS. One hundred ninety strains (179 clinical isolates previously identified by sequencing and the 11 reference strains), cultured on solid medium, were blindy analyzed by the MALDI-TOF MS technology to validate the generated in-house reference database. A 100% correlation was obtained with both identification methods, gene sequencing and MALDI-TOF MS, and no discordant identification was obtained. The HUVR database provided species level (score of ≥2.0) identification in 165 isolates (86.84%) and for the remaining 25 (13.16%) a genus level identification (score between 1.7 and 2.0) was obtained. The routine MALDI-TOF MS analysis with the new database, was then challenged with 200 Aspergillus clinical isolates grown on solid medium in a prospective evaluation. A species identification was obtained in 191 strains (95.5%), and only nine strains (4.5%) could not be identified at the species level. Among the 200 strains, A. tubingensis was the only cryptic species identified. We demonstrated the feasibility and usefulness of the new HUVR database in MALDI-TOF MS by the use of a standardized procedure for the identification of Aspergillus clinical isolates, including cryptic species, grown either on solid or liquid media.

Background: The appearance and dissemination of MDR among pathogenic bacteria has forced the search for new antimicrobials. Bacteriocins have been proposed as potential alternatives for the treatment of infections due to multiresistant strains., Objectives: To analyse the activity of R-pyocins against clinical isolates of Pseudomonas aeruginosa from patients with cystic fibrosis and other sources and evaluate them as a potential adjuvant or alternative to the current antibiotic treatment., Methods: The activity of R-pyocins against 150 strains of P. aeruginosa isolated from patients with cystic fibrosis or bacteraemia was studied through spot assay. Interactions between R-pyocins and antipseudomonal agents were quantitatively studied by the chequerboard method., Results: The proportion of P. aeruginosa isolates susceptible to R-pyocins was found to be higher in cystic fibrosis isolates compared with bacteraemia isolates (79.41% versus 50%). Moreover, no interactions were found between common antipseudomonal agents and R-pyocin susceptibility, except for the ST175 high-risk clone., Conclusions: Our results highlight the possibility of using R-pyocins as therapeutic agents, alone or as adjuvants, against P. aeruginosa in cystic fibrosis.

DGAT1 enzymes (acyl-CoA:diacylglycerol acyltransferase 1, EC 2.3.1.20) catalyse the formation of triacylglycerols (TAGs), the most abundant lipids in vegetable oils. Thorough understanding of the enzymology of oil accumulation is critical to the goal of modifying oilseeds for improved vegetable oil production. Four isoforms of BnDGAT1, the final and rate-limiting step in triacylglycerol synthesis, were characterized from Brassica napus, one of the world's most important oilseed crops. Transcriptional profiling of developing B. napus seeds indicated two genes, BnDGAT1-1 and BnDGAT1-2, with high expression and two, BnDGAT1-3 and BnDGAT1-4, with low expression. The activities of each BnDGAT1 isozyme were characterized following expression in a strain of yeast deficient in TAG synthesis. TAG from B. napus seeds contain only 10% palmitic acid (16:0) at the sn-3 position, so it was surprising that all four BnDGAT1 isozymes exhibited strong (4- to 7-fold) specificity for 16:0 over oleic acid (18:1) as the acyl-CoA substrate. However, the ratio of 18:1-CoA to 16:0-CoA in B. napus seeds during the peak period of TAG synthesis is 3:1. When substrate selectivity assays were conducted with 18:1-CoA and 16:0-CoA in a 3:1 ratio, the four isozymes incorporated 18:1 in amounts 2- to 5-fold higher than 16:0. This strong sensitivity of the BnDGAT1 isozymes to the relative concentrations of acyl-CoA substrates substantially explains the observed fatty acid composition of B. napus seed oil. Understanding these enzymes that are critical for triacylglycerol synthesis will facilitate genetic and biotechnological manipulations to improve this oilseed crop., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Objectives: To model the standard broth microdilution method, based on a modified Gompertz function, to obtain accurate vancomycin MIC values for methicillin-resistant Staphylococcus aureus (MRSA). The effect of these MIC values on the vancomycin therapeutic target of AUC(0-24)/MIC ≥ 400 was evaluated., Methods: Three clinical isolates of MRSA with different vancomycin MIC values were used in this model. The optical densities (OD) of each MIC determination were modelled by a non-linear regression method using an F-test. The OD data were adjusted to the Gompertz equation to obtain the MIC values. The mean vancomycin AUC(0-24) obtained with a 30 mg/kg/day dosing schedule was calculated using a Monte Carlo simulation over 5000 subjects, using the pharmacokinetic data obtained in vancomycin-treated patients in our hospital., Results: Although the MIC values obtained with this model were lower than those of the diffusion method (Etest) in all three cases, this did not affect the AUC(0-24)/MIC ratio for the strains with MICs of 1 mg/L by Etest. However, in those strains with MIC values >1 mg/L, the confidence intervals obtained for this ratio included values <400., Conclusions: The inherent variability of the broth microdilution method could explain the differences in the clinical outcome in MRSA-infected patients treated with vancomycin, mainly in those due to strains with MIC values of 1.5-2 mg/L by Etest, because the corresponding MIC values would range from 0.84 to 1.52 mg/L by the microdilution method, which could affect the therapeutic target.

Background: The plasminogen activator (PA) and matrix metalloproteinase (MMP) systems are implicated in the establishment of endometriosis. The mechanisms by which these systems are involved in the pathogenesis of this disease are not well defined and controversial results have been published. The aim of this study was to analyse mRNA and protein levels of several components of the PA and MMP systems in endometriotic tissue and endometrium from women with and without endometriosis., Methods and Results: Real-time quantitative RT-PCR assays were developed to quantify mRNA levels of these components in 57 women with endometriosis and 32 controls. Endometrium of women with endometriosis showed higher mRNA and antigenic levels of urokinase type-PA (uPA) and MMP-3 than endometrium from controls. In these patients, ovarian endometriotic tissue had higher mRNA and antigenic levels of PA inhibitor type 1 (PAI-1) and MMP inhibitor type 1 (TIMP-1) than endometrium., Conclusions: The increase in mRNA and protein levels of uPA and MMP-3 observed in endometrium of women with endometriosis may facilitate the attachment of endometrial tissue to the peritoneum and ovarian surface, as well as the invasion of the extracellular matrix. This process would lead to the formation of early endometriotic lesions. Once the ovarian endometriotic cyst is developed, PAI-1 and TIMP-1 would increase which could explain the frequent clinical finding of an endometrioma without invasion of the adjacent ovarian tissue.

Background: Hypertensive patients show greater platelet activation than do normotensive individuals. Platelet activation is characterized by increased phosphatidylserine (PS) exposure in the external hemilayer of the membrane, a larger number of platelet microparticles (PMP), and changes in intraplatelet-free calcium kinetics. This study evaluated whether eprosartan can protect against undesirable platelet activation., Methods: A total of 30 hypertensive patients (systolic blood pressure [SBP] 140 to 189 mm Hg; diastolic blood pressure [DBP] 90 to 109 mm Hg) without renal, liver, or cardiac organic lesions and with a mean age of 47.6 +/- 9.4 years and mean body mass index (BMI) of 27.9 +/- 3.9 kg/m2 received eprosartan (600 mg/day). They were compared with 31 normotensive individuals with a mean age of 43.3 +/- 6.7 years and a mean BMI of 26.8 +/- 3.9 kg/m2. Blood pressure measurements and platelet function changes were assessed at baseline (control and hypertensive patients) and after 1 and 2 months of eprosartan monotherapy (hypertensive patients only)., Results: Significant baseline to endpoint (month 2) changes in SBP and DBP were noted in the eprosartan group (SBP: baseline 152.2 +/- 16.8 mm Hg, endpoint 142.2 +/- 16.9 mm Hg, P <.01; DBP: baseline 93.5 +/- 9.9 mm Hg, endpoint 85.8 +/- 11.9 mm Hg, P <.001). Native circulating activated platelets increased in both groups after shear stress or Ca2+ ionophore activation, and were reduced by eprosartan (after shear exposure from 104% at month 1 to 76% after 2 months of therapy). Eprosartan therapy normalized the number of microparticles after blood shear exposure (P <.01) and after exposure to Ca2+ ionophore activation (P <.05) and significantly reduced the trend for platelets to be more readily activated in hypertensive compared with normotensive subjects (baseline to endpoint change P <.001; increase/shear versus baseline P <.001). Eprosartan partially normalizes cytoplasmic-free calcium mobilization in platelets., Conclusions: Eprosartan significantly reduces blood pressure and normalizes undesirable changes in platelet function., (Copyright 2004 American Journal of Hypertension, Ltd.)

Background: Endometriosis is considered a benign disease that has the ability to invade normal tissue. As in neoplastic growth, local extracellular proteolysis may take place. The aim of this study is to analyse several components of the plasminogen activator (PA) pathway and the matrix metalloproteinase (MMP) system in endometriotic tissue, endometrium and peritoneal fluid from women with and without endometriosis (controls)., Methods and Results: Thirty-nine women with endometriosis and 35 controls were studied. In eutopic endometrium of women with endometriosis, the antigenic levels of urokinase-type PA (uPA) and MMP-3 were elevated when compared with endometrium from controls. Ovarian endometriotic tissues had higher antigenic levels of PA inhibitor type 1 (PAI-1) and tissue inhibitor of metalloproteinases type 1 (TIMP-1) than endometrium. The peritoneal fluid from women with endometriosis showed a significant increase in uPA levels compared with controls., Conclusions: The increase in antigenic levels of uPA and MMP-3 in endometrium of women with endometriosis might contribute to the invasive potential of endometrial cells. Once the ovarian endometriotic cyst is developed, an increase in PAI-1 and TIMP-1 is detected and significant proteolytic activity is no longer observed. This increase in inhibitors and decrease in proteolytic activity could explain the frequent clinical finding of isolated endometriotic cyst without invasion of the surrounding ovarian tissue.

Background: The plasminogen activation system and matrix metalloproteinases (MMPs) play a key role in the degradation of basement membrane and extracellular matrix in tissue remodeling, cancer cell invasion, and metastasis., Methods: Quantitative real-time reverse-transcription-PCR (RT-PCR) assays were developed to quantify urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor type 1 (PAI-1), and tissue metalloproteinase inhibitor type 1 (TIMP-1) mRNA in 54 breast cancer tissues. Gene fragments were amplified in a LightCycler real-time PCR system using gene-specific primers and SYBR Green I. The results were normalized to beta-actin mRNA. We also quantified antigen and functional concentrations of these components., Results: The intra- and interassay variabilities for mRNA quantification showed mean SDs for the crossing point of 0.12 and 0.15 cycles, respectively. PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and PAI-1 and uPA functional concentrations increased with tumor severity; the increase was statistically significant for PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and for uPA functional concentrations. Node-positive patients showed significantly higher PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations than those who were node negative., Conclusions: Quantitative real-time RT-PCR is a highly sensitive, reproducible, and fast method for measuring gene expression of PAI-1, uPA, and TIMP-1 in breast cancer. These components may be involved in breast cancer development, and increased mRNA expression may be associated with a worse prognosis.

Background: The aim of this study was to assess the diagnostic accuracy of the proportion of prostate-specific antigen (PSA) complexed to alpha(1)-antichymotrypsin (PSA-alpha(1)ACT:PSA ratio) in the differential diagnosis of prostate cancer (CaP) and benign prostatic hyperplasia (BPH) in men with total PSA of 10-30 microg/L., Methods: We used our immunoassays (ELISAs) for total PSA and PSA-alpha(1)ACT complex to study 146 men. In 123, total PSA was between 10 and 20 microg/L; 66 of these had CaP and 57 BPH. In 23 men, total PSA was between 20 and 30 microg/L; 14 of these had CaP and 9 BPH. We calculated the area under the ROC curves (AUC) for total PSA, PSA-alpha(1)ACT complex, and PSA-alpha(1)ACT:PSA ratio, and determined the cutoff points that gave sensitivities approaching 100%., Results: In the total PSA range between 10 and 20 microg/L, the AUC was significantly higher for the PSA-alpha(1)ACT:PSA ratio (0.850) than for total PSA (0.507) and PSA-alpha(1)ACT complex (0.710; P <0.0001). A cutoff ratio of 0.62 would have permitted diagnosis of all 66 patients with CaP (100% sensitivity) and avoided 19% of unnecessary biopsies (11 of 57 patients). In the total PSA range between 20 and 30 microg/L, the AUC for the PSA-alpha(1)ACT:PSA ratio (0.980; 95% confidence interval, 0.82-0.99) was greater than the AUC for total PSA (0.750; 95% confidence interval, 0.51-0.89; P = 0.042). In this range, a cutoff point of 0.64 would have permitted the correct diagnosis of all 14 patients with CaP and 6 of the 9 with BPH., Conclusions: The diagnostic accuracy of the PSA-alpha(1)ACT:PSA ratio persists at high total PSA concentrations, increasing the specificity of total PSA. Prospective studies with large numbers of patients are needed to assess whether the ratio of PSA-alpha(1)ACT to total PSA is a useful tool to avoid unnecessary prostatic biopsy in patients with a total PSA >10 microg/L.

Background: The aim of the present study was to assess the antialbuminuric effect of losartan in a large number of hypertensive type 2 diabetics., Methods: This was a 6-month, open-label, prospective and multicentre study. A total of 422 patients with type 2 diabetes who were hypertensive [sitting systolic blood pressure (SBP) > or = 140 mmHg and/or diastolic blood pressure (DBP) > or = 90 mmHg] and microalbuminuric [urinary albumin excretion (UAE) 30-300 mg/day] were eligible for the study. After a 2-week run-in period, patients were placed on losartan 50 mg once a day. If the BP did not reach the desired goal (< 140/90 mmHg) after a 4-week period, the losartan dose was doubled. In the absence of control of BP, losartan 50 mg/day+hydroclorothiazide 12.5 mg/day was administrated. Initially and at 12 and 24 weeks of active treatment, BP, UAE, HbA(1c) and other renal function parameters were evaluated., Results: A significant decrease in SBP and DBP was observed, as well as in parameters reflecting metabolic control, fasting glucose and HbA(1c). UAE also decreased significantly, but the percentage of the variance of change in UAE explained by the changes in SBP and HbA(1c) was, however, negligible, i.e. 4%. Moreover, small but significant reductions in uric acid, total cholesterol and triglycerides, and an increase in HDL-cholesterol levels were also observed., Conclusion: Antihypertensive treatment with losartan exerts a beneficial effect on UAE, a benchmark for measuring the efficacy of therapeutic interventions in diabetic nephropathy, by reducing BP and allowing better diabetes control. The role of other mechanisms influencing the favourable outcome, beyond these measured effects, needs to be assessed in further studies.

The in-vitro activity of sparfloxacin against four pathogens commonly implicated in genital infections was compared with that of a number of other commonly administered antimicrobials. Sparfloxacin demonstrated excellent activity against Neisseria gonorrhoeae (MIC range of < or = 0.0002-5 mg/L for beta-lactamase producing strains, and < or = 0.0002-0.03 mg/L for non-beta-lactamase producing strains). This activity was similar to that of lomefloxacin and ciprofloxacin and was greater than that of ofloxacin. Sparfloxacin was more active against Ureaplasma urealyticum (MIC90 1 mg/L) than the other three quinolones (MIC90 4 mg/L). Sparfloxacin was much more active against Mycoplasma hominis (MIC90 0.06 mg/L) than the other quinolones (MIC90 1 mg/L). Sparfloxacin showed the most potent inhibitory and bactericidal activity of the quinolones against Chlamydia trachomatis with MIC and MBC of 0.06 mg/L (ofloxacin MIC and MBC 1 mg/L; ciprofloxacin and lomefloxacin MIC and MBC 2 mg/L). The results of this study and others performed by workers using different methods are consistently similar. Since sparfloxacin has broad activity against pathogens implicated in genital infections it may be a good therapeutic alternative for these syndromes.

Uraemic patients suffer from haemorrhagic disorders and accelerated atherosclerosis. To evaluate the possible role of the vessel wall in these haemostatic alterations associated with uraemia, we investigated the effect of a uraemic milieu on human endothelial cell (EC) cultures and the reactivity of the extracellular matrices (ECM) generated by these cells towards platelets. EC cultures were exposed to a pool of sera (20% in the culture medium) obtained either from uraemic patients or from normal donors, and the following parameters were evaluated: (1) EC viability (trypan blue exclusion test); (2) von Willebrand factor (vWF) levels in supernatants and associated with ECM; (3) the reactivity of EC and EC-derived ECM towards platelets, measured 'ex vivo' under flow conditions (5 min, wall shear rate 800 s-1); and (4) ultrastructure of the ECM. The viability of EC cultured in the presence of uraemic sera was similar to controls. Platelet interaction with ECM generated by EC exposed to uraemic sera was significantly reduced (P < 0.05). This decrease was mainly related to a reduction in platelet adhesion (9.8 +/- 1.9% vs 16.7 +/- 1.8% in controls, P < 0.02). VWF levels in supernatants and associated with ECM were similar to controls. Ultrastructural analysis of the ECM generated by EC exposed to uraemic sera revealed a deficient matrix. An increased removal of EC was observed in experiments in which EC cultured in the presence of uraemic sera were perfused with citrated blood. These results indicate that a uraemic milieu induces quantitative and qualitative changes in the vascular subendothelium, characterized by a less intrincate network of fibrils, as well as a decreased attachment of EC and reduced thrombogenicity to the ECM. These changes may represent another mechanism which contributes to the haemostatic dysfunction observed in uraemic patients.

Here we summarize the recent progress in research on the role of the fibrinolytic system in reproduction, with a special emphasis on the role of the plasminogen activator inhibitors in fetal development. Trophoblasts produce fibrinolytic proteins that can promote normal implantation and regulate blood flow to the fetus and placenta throughout pregnancy. Normal pregnancy is associated with a hypofibrinolytic state that is fundamentally caused by an increase in plasminogen activator inhibitors types 1 and 2. In pre-eclampsia, a fibrinolytic failure, resulting from an increase in plasma and placental concentrations of plasminogen activator inhibitor-1, was observed. The localized elevated concentrations of placenta plasminogen activator inhibitor-1 protein and mRNA observed in pre-eclamptic patients would be expected to foster the deposition of fibrin and thus play a role in the complications associated with this disease. The decreased plasminogen activator inhibitor-2 concentrations in placenta and plasma from intrauterine fetal growth retardation pregnancies and the positive correlation between plasma/placenta plasminogen activator inhibitor-2 concentration and birthweight suggest that this inhibitor could be considered an adequate marker of placental function.

Activity of imipenem (N-formimidoyl thienamycin, ampicillin and vancomycin against 27 Streptococcus faecalis and 5 Str. faecium was determined. Also evaluated were the interactions of imipenem, ampicillin and vancomycin with streptomycin or amikacin or sissomicin. Imipenem showed the most inhibitory activity against Str. faecalis with MIC50 and MIC90 values 0.5 and 2 mg/l respectively, Str. faecium strains were less susceptible to imipenem with MIC50/MIC90 values of 2/16 mg/l. Imipenem showed good bactericidal activity against both groups of strains with MBC50/MBC90 values of 1/4 and 4/16 mg/l respectively. The most favourable interactions (synergy and addition) of imipenem were with streptomycin (72.2%) and sissomicin (53.2%). Combinations containing amikacin were the least favourable. Similar results were obtained with these combinations against Str. faecium.