Tryptophan Fluorescence Quenching in β-Lactam-Interacting Proteins Is Modulated by the Structure of Intermediates and Final Products of the Acylation Reaction

In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of d,d-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by l,d-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model l,d-transpeptidase (Ldt) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldt. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation

The prognosis of streptococcal prosthetic bone and joint infections depends on surgical management-A multicenter retrospective study

BACKGROUND: The optimal treatment of streptococcal prosthetic joint infections (PJIs) is unclear. METHODS: A cohort of streptococcal PJIs was reviewed retrospectively in seven reference centers for the management of complex bone and joint infections, covering the period January 1, 2010 to December 31, 2012. RESULTS: Seventy patients with monomicrobial infections were included: 47 had infections of total hip arthroplasty and 23 had infections of total knee arthroplasty. The median age was 77 years (interquartile range (IQR) 69-83 years), the median Charlson comorbidity score was 4 (IQR 3-6), and 15.6% (n=11) had diabetes. The most commonly identified streptococcal species were Streptococcus agalactiae and Streptococcus dysgalactiae (38.6% (n=27) and 17.1% (n=12), respectively). Debridement, antibiotics and implant retention (DAIR) was performed after a median time of 7 days (IQR 3-8 days), with polyethylene exchange (PE) in 21% of cases. After a minimum follow-up of 2 years, 27% of patients had relapsed, corresponding to 51.4% of DAIR treatment cases and 0% of one-stage (n=15) or two-stage (n=17) exchange strategy cases. Rifampicin or levofloxacin in combination therapy was not associated with a better outcome (adjusted p= 0.99). S. agalactiae species and DAIR treatment were associated with a higher risk of failure. On multivariate analysis, only DAIR treatment and S. agalactiae were independent factors of relapse. Compared to DAIR without PE, DAIR with PE was only associated with a trend towards a benefit (odds ratio 0.33, 95% confidence interval 0.06-1.96; adjusted p= 0.44). CONCLUSIONS: Streptococcal PJIs managed with DAIR have a poor prognosis and S. agalactiae seems to be an independent factor of treatment failure

Tissue perfusion alterations correlate with mortality in patients admitted to the intensive care unit for acute pulmonary embolism

We aimed to assess the relationship between alterations of tissue perfusion parameters at admission (highly predictive of mortality in septic shock) and outcome in patients admitted to the intensive care unit (ICU) for acute pulmonary embolism (PE). We conducted a retrospective study to analyze the association between arterial lactate level, skin mottling and urinary output, and 28-day mortality. Over a 22-year period, 317 patients with PE were identified but we finally analyzed 108 patients whose main diagnosis for ICU admission was acute PE. At admission, the sequential organ failure assessment score was 2 (0–6) and the simplified acute physiology score II was 29 (16–43). Thirty patients (28%) received vasopressors and 37 patients (34%) received thrombolytic therapy. Day 28 mortality rate was 25% (n = 27). When compared to 28-day survivors, nonsurvivor patients had higher lactate level (4.5 [2.3–10.3] mmol/L vs 1.4 [1–2.9] mmol/L, P < .0001), more frequent mottling around the knee area (56% vs 25%, P = .003) and a lower urinary output (during the first 6 hours) (0.35 [0–1] mL/kg/h vs. 0.88 [0.62–1.677] mL/kg/h, P = .0002). Mortality increased with the number of tissue perfusion alterations present upon admission, 8% for none, 21% for 1, 28% for 2, and finally reached 85% for 3 tissue perfusion alterations (P < .0001). In a multivariate analysis, the relationship between the number of tissue perfusion alterations and 28-day mortality was maintained after adjustment on the presence of shock and right ventricular dilation at admission. In ICU patients admitted for acute PE, tissue perfusion alterations correlated with 28-day mortality independently of blood pressure and right ventricular dilation

The Weaning Index combining EtCO2 and respiratory rate early identifies Spontaneous Breathing trial failure. A pilot study

BACKGROUND: We aimed to evaluate the predictive value of the end-tidal CO2 (EtCO2) alone or combined with ventilation related parameters on spontaneous breathing trial (SBT) outcome on mechanically ventilated patients. METHODS: Prospective observational study in a medical ICU. Mechanically ventilated adult patients who met predefined criteria for weaning were included. Patients underwent a T-piece SBT for 30 minutes and the usual hemodynamic and respiratory clinical parameters including EtCO2 were recorded every 5 minutes. RESULTS: 280 patients were studied (age: 64±17 years, SAPS II: 44 [34-56]) during a first SBT and 76 patients during a second SBT. The Weaning Index, defined as the product of the respiratory rate and EtCO2, was a strong early predictive factor of SBT outcome; at 10 minutes, the area under the curve (AUC) was 86% ([80-90], P<0.0001) during the first SBT and 88% ([80-96], P<0.0001) during the second SBT. After 10 minutes of SBT, a Weaning Index >1100 identified patients that will not successfully complete the SBT at 30 minutes with a specificity of 98%. CONCLUSIONS: In unselected mechanically ventilated patients, the Weaning Index is helpful to early identify patients who will fail the SBT during a first and a second trial

Reversible Microvascular Hyporeactivity to Acetylcholine During Diabetic Ketoacidosis

OBJECTIVES: Metabolic acidosis is commonly observed in critically ill patients. Experimental studies suggested that acidosis by itself could impair vascular function, but this has been poorly investigated in human. DESIGN: Prospective observational study. SETTING: Medical ICU in a tertiary teaching hospital. PATIENTS: To assess the relationship between metabolic acidosis severity and microvascular reactivity, we included adult diabetic patients admitted in ICU for ketoacidosis. Microvascular response to acetylcholine iontophoresis was measured at admission (baseline) and after correction of metabolic acidosis (24 hr). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Thirty-nine patients with diabetic ketoacidosis were included (68% male), with a median age of 43 (31-57) years. At admission, microvascular reactivity negatively correlated with acidosis severity (R = -0.53; p < 0.001). Microvascular response was strongly depressed at pH less than 7.20 (area under the curve, 1,779 [740-3,079] vs 12,944 [4,874-21,596] at pH > 7.20; p < 0.0001). In addition, acidosis severity was significantly correlated with capillary refill time (R = 0.50; p = 0.02). At H24, after rehydration and insulin infusion, clinical and biological disorders were fully corrected. After acidosis correction, microvascular reactivity increased more in patients with severe baseline acidosis (pH < 7.20) than in those with mild baseline acidosis (area under the curve, +453% [213%-1,470%] vs +121% [79%-312%]; p < 0.01). CONCLUSIONS: We identified an alteration of microvascular reactivity during metabolic acidosis in critically ill patients with diabetic ketoacidosis. Microvascular hyporeactivity recovered after acidosis correction