Study of the cwaRS-ldcA Operon Coding a Two-Component System and a Putative L,D-Carboxypeptidase in Lactobacillus paracasei.

The cell surface is the primary recognition site between the bacterium and the host. An operon of three genes, LSEI_0219 (cwaR), LSEI_0220 (cwaS), and LSEI_0221 (ldcA), has been previously identified as required for the establishment of Lactobacillus paracasei in the gut. The genes cwaR and cwaS encode a predicted two-component system (TCS) and ldcA a predicted D-alanyl-D-alanine carboxypeptidase which is a peptidoglycan (PG) biosynthesis enzyme. We explored the functionality and the physiological role of these three genes, particularly their impact on the bacterial cell wall architecture and on the bacterial adaptation to environmental perturbations in the gut. The functionality of CwaS/R proteins as a TCS has been demonstrated by biochemical analysis. It is involved in the transcriptional regulation of several genes of the PG biosynthesis. Analysis of the muropeptides of PG in mutants allowed us to re-annotate LSEI_0221 as a putative L,D-carboxypeptidase (LdcA). The absence of this protein coincided with a decrease of two surface antigens: LSEI_0020, corresponding to p40 or msp2 whose implication in the host epithelial homeostasis has been recently studied, and LSEI_2029 which has never been functionally characterized. The inactivation of each of these three genes induces susceptibility to antimicrobial peptides (hBD1, hBD2, and CCL20), which could be the main cause of the gut establishment deficiency. Thus, this operon is necessary for the presence of two surface antigens and for a suitable cell wall architecture.

Identification and transcriptional profile of Lactobacillus paracasei genes involved in the response to desiccation and rehydration.

Lactobacillus paracasei is able to persist in a variety of natural and technological environments despite physico-chemical perturbations, in particular alternations between desiccation and rehydration. However, the way in which it adapts to hydric fluctuations and the genetic determinants involved are not clearly understood. To identify the genes involved in adaptation to desiccation, an annotated library of L. paracasei random transposon mutants was screened for viability after desiccation (25% relative humidity, 25 °C). We found 16 genes that have not been described as being involved in this response. Most of them are linked to either the transport of molecules or to cell wall structure and function. Our screening also identified genes encoding DNA related enzymes and an alarmone necessary for L. paracasei survival. Subsequently, the expression of the identified genes was measured at five stages of the dehydration-rehydration process to decipher the chronology of genetic mechanisms. They were classified into four different transcriptional profiles: genes upregulated during both desiccation and rehydration phases, genes upregulated during the desiccation phase only, genes downregulated during both desiccation and rehydration and genes downregulated only during the rehydration stage. Thus, genetic response to hydric fluctuations seems to occur during desiccation and can continue or not during rehydration. The genes identified should contribute to improve the stabilization of Lactobacillus starters in dry state.

Impact of subglottic secretion drainage on microaspiration in critically ill patients: a prospective observational study.

BACKGROUND: Microaspiration is a major factor in ventilator-associated pneumonia (VAP) pathophysiology. Subglottic secretion drainage (SSD) aims at reducing its incidence. METHODS: Single-center prospective observational study, performed in a French intensive care unit (ICU) from March 2012 to April 2013, including adult patients mechanically ventilated for at least 24 hours divided in two groups: patients in the SSD group intubated using tracheal tubes allowing SSD and patients in the control group intubated with standard tracheal tubes. Pepsin and salivary amylase concentrations were measured for 24 hours in all tracheal aspirates. Primary objective was to determine the impact of SSD on gastric or oropharyngeal microaspiration using pepsin or amylase concentration in tracheal aspirates. RESULTS: Fifty-five patients were included in the SSD group and 45 in the control group. No difference was found between groups regarding the incidence of microaspiration defined as at least one tracheal aspirate positive for either pepsin or amylase [49 (89%) vs. 37 (82%), P=0.469]. Percentage of patients with VAP [16 (29%) vs. 11 (24%), P=0.656], ventilator-associated tracheobronchitis (VAT) [7 (13%) vs. 4 (9%), P=0.750] or early airway colonization [15 (35%) vs. 8 (18%), P=0.219] were not significantly different in study groups. CONCLUSIONS: SSD did not reduce the incidence of microaspiration, VAP, VAT or airway colonization in this observational study.

New Genes Involved in Mild Stress Response Identified by Transposon Mutagenesis in Lactobacillus paracasei.

Lactic acid bacteria (LAB) are associated with various plant, animal, and human niches and are also present in many fermented foods and beverages. Thus, they are subjected to several stress conditions and have developed advanced response mechanisms to resist, adapt, and grow. This work aimed to identify the genes involved in some stress adaptation mechanisms in LAB. For this purpose, global reverse genetics was applied by screening a library of 1287 Lactobacillus paracasei transposon mutants for mild monofactorial stresses. This library was submitted independently to heat (52°C, 30 min), ethanol (170 g.L-1, 30 min), salt (NaCl 0.8 M, 24 h), acid (pH 4.5, 24 h), and oxidative (2 mM H2O2, 24 h) perturbations which trigger mild monofactorial stresses compatible with bacterial adaptation. Stress sensitivity of mutants was determined either by evaluating viability using propidium iodide (PI) staining, or by following growth inhibition through turbidity measurement. The screening for heat and ethanol stresses lead respectively to the identification of 63 and 27 genes/putative promoters whose disruption lead to an increased sensitivity. Among them, 14 genes or putative promoters were common for both stresses. For salt, acid and oxidative stresses, respectively 8, 6, and 9 genes or putative promoters were identified as essential for adaptation to these unfavorable conditions, with only three genes common to at least two stresses. Then, RT-qPCR was performed on selected stress response genes identified by mutant screenings in order to evaluate if their expression was modified in response to stresses in the parental strain. Eleven genes (membrane, transposase, chaperone, nucleotide and carbohydrate metabolism, and hypothetical protein genes) were upregulated during stress adaptation for at least two stresses. Seven genes, encoding membrane functions, were upregulated in response to a specific stress and thus could represent potential transcriptomic biomarkers. The results highlights that most of the genes identified by global reverse genetics are specifically required in response to one stress and that they are not differentially transcribed during stress in the parental strain. Most of these genes have not been characterized as stress response genes and provide new insights into the adaptation of lactic acid bacteria to their environment.

Evaluation of endothelial biomarkers as predictors of organ failures in septic shock patients.

BACKGROUND: Endothelial injury is recognized to trigger organ failures during the first 48h of septic shock. We evaluate endothelial biomarkers at ICU admission in their ability to predict severity, outcome, and organ failures in septic shock patients. METHODS: This prospective observational pilot study was conducted in a medical intensive care unit of a university hospital. Plasma levels of endothelial biomarkers as angiopoietin-2, sE-selectin or endocan were measured at ICU admission of 20 patients presenting with septic shock. Clinical and biological data were recorded at inclusion and each day during the first week. RESULTS: Significant correlations were found between angiopoietin-2 and severity scores at Day 1: SAPS2 (r(2)=0.620; p=0.004) and LOD score (r(2)=0.681; p=0.001). The angiopoietin-2 level was significantly higher in patients presenting with organ failure such as hemodynamic, renal or hepatic failure. It correlated with catecholamine infusion dose and was higher in non survivors compared with survivors (33.5 [28.9-51.4] vs. 12.4 [6.4-14.7]ng/ml; p=0.001). In contrast, in that population presenting with septic shock, endocan level at inclusion was not related to any organ failure at inclusion or Day 1 but appeared lower in patients presenting with respiratory failure at Day 3 compared to those who do not (1.9 [0.99-3.1] vs 5.2 [3.1-17.2]ng/ml; p=0.032). The endocan level at inclusion was correlated with the decrease in PaO2/FiO2 ratio at Day 2 (r(2)=0.628; p=0.0004) and Day 3 (r(2)=0.645; p=0.005). Endocan level <2.54ng/ml at admission is predictive of a respiratory failure presence at Day 3. CONCLUSION: In septic shock patients, angiopoietine-2 is related with clinical severity during the first 24h but only endocan is able to predict the presence of respiratory failure at Day 3.

Macrophage migration inhibitory factor inhibition is deleterious for high-fat diet-induced cardiac dysfunction.

AIMS: Development of metabolic syndrome is associated with impaired cardiac performance, mitochondrial dysfunction and pro-inflammatory cytokine increase, such as the macrophage migration inhibitory factor MIF. Depending on conditions, MIF may exert both beneficial and deleterious effects on the myocardium. Therefore, we tested whether pharmacological inhibition of MIF prevented or worsened metabolic syndrome-induced myocardial dysfunction. METHODS AND RESULTS: C57BL/6J mice were fed for ten weeks with 60% fat-enriched diet (HFD) or normal diet (ND). MIF inhibition was obtained by injecting mice twice a week with ISO-1, for three consecutive weeks. Then, triglycerides, cholesterol, fat mass, glucose intolerance, insulin resistance, ex vivo cardiac contractility, animal energetic substrate utilization assessed by indirect calorimetry and mitochondrial respiration and biogenesis were evaluated. HFD led to fat mass increase, dyslipidemia, glucose intolerance and insulin resistance. ISO-1 did not alter these parameters. However, MIF inhibition was responsible for HFD-induced cardiac dysfunction worsening. Mouse capacity to increase oxygen consumption in response to exercise was reduced in HFD compared to ND, and further diminished in ISO-1-treated HFD group. Mitochondrial respiration was reduced in HFD mice, treated or not with ISO-1. Compared to ND, mitochondrial biogenesis signaling was upregulated in the HFD as demonstrated by mitochondrial DNA amount and PGC-1α expression. However, this increase in biogenesis was blocked by ISO-1 treatment. CONCLUSION: MIF inhibition achieved by ISO-1 was responsible for a reduction in HFD-induced mitochondrial biogenesis signaling that could explain majored cardiac dysfunction observed in HFD mice treated with MIF inhibitor.

Unintentional strangulation by a cervical collar after attempted suicide by hanging.

We report the case of a young man who attempted suicide by hanging and whose neurological status deteriorated until the cervical collar, that had been correctly placed by the prehospital team, was removed. We discuss the physiopathological mechanisms leading to death in hanging that is, a blockage of the blood stream to the brain leading to vasogenic and cytotoxic cerebral edema rather than asphyxia or spinal fracture. Our case supports the early removal of neck stabilization devices that can dangerously harm the patient after an attempted suicide by hanging, by increasing intracerebral pressure.