Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10(-5) on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Antibiotic resistance of heterotrophic bacteria in sewage-contaminated rivers

info:eu-repo/semantics/nonPublishe

Identification of a metagenomic gene cluster containing a new class A beta-lactamase and toxin-antitoxin systems.

Several reports mention the presence of antibiotic resistance genes in natural and polluted environments, but many studies are based on their detection via polymerase chain reaction (PCR amplification of known genes and not on an activity screening. We constructed a metagenomic fosmid bank from DNA isolated from a polluted river in Brussels, Belgium, the Zenne. A total of 120,000 clones were pooled and plated directly on solid media containing different antibiotics. Several clones were isolated which could grow in the presence of ampicillin. The DNA from several clones was extracted and subjected to restriction analysis and, based on their restriction pattern, two different clones were found. One of the clones was selected for further study as it showed a higher level of resistance to different β-lactams antibiotics (ticarcilline and ceftazidime). To find out which gene is responsible for the resistance, an in vitro transposon mutagenesis was performed and clones having lost the resistance phenotype were analyzed via inverse PCR amplification. Several clones had an insert in a gene encoding a new type of β-lactamase. The amplified fosmid DNA was fully sequenced revealing an insert of 41 kb containing 39 open reading frames (ORFs). Transposon insertions inactivating the resistance to β-lactams were also found in the ORF upstream of the blaA gene, encoding an aminotransferase, suggesting a polar effect on the transcription of the gene downstream. In addition, other genes were found such as histidine biosynthesis genes, which were found to be scattered on the insert, a relA/spoT gene, and genes belonging to type II toxin-antitoxin system. This predicted system was experimentally validated in Escherichia coli using an inducible expression system.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

The skin microbiome of caspase-14-deficient mice shows mild dysbiosis

Caspase-14, an important proteinase involved in filaggrin catabolism, is mainly active in terminally differentiating keratinocytes, where it is required for the generation of skin natural moisturizing factors (NMFs). Consequently, caspase-14 deficient epidermis is characterized by reduced levels of NMFs such as urocanic acid and 2-pyrrolidone-5-carboxylic acid. Patients suffering from filaggrin deficiency are prone to develop atopic dermatitis, which is accompanied with increased microbial burden. Among several reasons, this effect could be due to a decrease in filaggrin breakdown products. In this study, we found that caspase-14(-/-) mice show enhanced antibacterial response compared to wild-type mice when challenged with bacteria. Therefore, we compared the microbial communities between wild-type and caspase-14(-/-) mice by sequencing of bacterial 16S ribosomal RNA genes. We observed that caspase-14 ablation leads to an increase in bacterial richness and diversity during steadystate conditions. Although both wild-type and caspase-14(-/-) skin were dominated by the Firmicutes phylum, the Staphylococcaceae family was reduced in caspase-14(-/-) mice. Altogether, our data demonstrated that caspase-14 deficiency causes the imbalance of the skin-resident bacterial communities

<i>Pseudomonas aeruginosa</i> LysR PA4203 regulator NmoR acts as a repressor of the PA4202 <i>nmoA</i> gene, encoding a nitronate monooxygenase

The PA4203 gene encodes a LysR regulator and lies between the ppgL gene (PA4204), which encodes a periplasmic gluconolactonase, and, in the opposite orientation, the PA4202 (nmoA) gene, coding for a nitronate monooxygenase, and ddlA (PA4201), encoding a d-alanine alanine ligase. The intergenic regions between PA4203 and ppgL and between PA4203 and nmoA are very short (79 and 107 nucleotides, respectively). Here we show that PA4203 (nmoR) represses its own transcription and the expression of nmoA. A chromatin immunoprecipitation analysis showed the presence of a single NmoR binding site between nmoA and nmoR, which was confirmed by electrophoretic mobility shift assays (EMSAs) with the purified NmoR protein. Despite this observation, a transcriptome analysis revealed more genes to be affected in an nmoR mutant, including genes known to be part of the MexT LysR activator regulon. The PA1225 gene, encoding a quinone oxidoreductase, was the most highly upregulated gene in the nmoR deletion mutant, independently of MexT. Finally, deletion of the nmoA gene resulted in an increased sensitivity of the cells to 3-nitropropionic acid (3-NPA), confirming the role of the nitronate monooxygenase protein in the detoxification of nitronate

Phenotypic and genome-wide analysis of an antibiotic-resistant small colony variant (SCV) of Pseudomonas aeruginosa

status: publishe

Efficacy and safety of dapagliflozin in patients with inadequately controlled type 1 diabetes (DEPICT-1):24 week results from a multicentre, double-blind, phase 3, randomised controlled trial

Background Dapagliflozin is a sodium-glucose cotransporter-2 inhibitor approved for the treatment of type 2 diabetes. We aimed to assess the efficacy and safety of dapagliflozin as an add-on to adjustable insulin in patients with inadequately controlled type 1 diabetes. Methods DEPICT-1 was a double-blind, randomised, parallel-controlled, three-arm, phase 3, multicentre study done at 143 sites in 17 countries. Eligible patients were aged 18–75 years and had inadequately controlled type 1 diabetes (HbA1cbetween ≥7·7% and ≤11·0% [≥61·0 mmol/mol and ≤97·0 mmol/mol]) and had been prescribed insulin for at least 12 months before enrolment. After an 8 week lead-in period to optimise diabetes management, patients were randomly assigned (1:1:1) using an interactive voice response system to dapagliflozin 5 mg or 10 mg once daily, given orally, or matched placebo. Randomisation was stratified by current use of continuous glucose monitoring, method of insulin administration, and baseline HbA1c. The primary efficacy outcome was the change from baseline in HbA1cafter 24 weeks of treatment in the full analysis set, which consisted of all randomly assigned patients who received at least one dose of study drug. An additional 55 patients who were incorrectly and non-randomly allocated to only dapagliflozin treatment groups were included in the safety analysis set. This study was registered with ClinicalTrials.gov, number NCT02268214; data collection for the present analysis was completed on Jan 4, 2017, and a 28 week extension phase is ongoing. Findings Between Nov 11, 2014, and April 16, 2016, 833 patients were assigned to treatment groups and included in safety analyses (dapagliflozin 5 mg [n=277] vs dapagliflozin 10 mg [n=296] vs placebo [n=260]; 778 of these patients were randomly assigned and included in the full analysis set for efficacy analyses (259 vs 259 vs 260; difference due to randomisation error affecting 55 patients). Mean baseline HbA1cwas 8·53% (70 mmol/mol; SD 0·67% [7·3 mmol/mol]). At week 24, both doses of dapagliflozin significantly reduced HbA1ccompared with placebo (mean difference from baseline to week 24 for dapagliflozin 5 mg vs placebo was −0·42% [95% CI −0·56 to −0·28; p&lt;0·0001] and for dapagliflozin 10 mg vs placebo was −0·45% [−0·58 to −0·31; p&lt;0·0001]). Among patients in the dapagliflozin 5 mg (n=277), dapagliflozin 10 mg (n=296), and placebo (n=260) groups, the most common adverse events were nasopharyngitis (38 [14%] vs 36 [12%] vs 39 [15%]), urinary tract infection (19 [7%] vs 11 [4%] vs 13 [5%]), upper respiratory tract infection (15 [5%] vs 15 [5%] vs 11 [4%]), and headache (12 [4%] vs 17 [6%] vs 11 [4%]). Hypoglycaemia occurred in 220 (79%), 235 (79%), and 207 (80%) patients in the dapagliflozin 5 mg, dapagliflozin 10 mg, and placebo groups, respectively; severe hypoglycaemia occurred in 21 (8%), 19 (6%), and 19 (7%) patients, respectively. Adjudicated definite diabetic ketoacidosis occurred in four (1%) patients in the dapagliflozin 5 mg group, five (2%) in the dapagliflozin 10 mg group, and three (1%) in the placebo group. Interpretation Our results suggest that dapagliflozin is a promising adjunct treatment to insulin to improve glycaemic control in patients with inadequately controlled type 1 diabetes. Funding AstraZeneca and Bristol-Myers Squibb