Stefaan J. Vandecasteele

Linezolid-induced inhibition of mitochondrial protein synthesis.

BACKGROUND Linezolid is an oxazolidinone antibiotic that is increasingly used to treat drug-resistant, gram-positive pathogens. The mechanism of action is inhibition of bacterial protein synthesis. Optic and/or peripheral neuropathy and lactic acidosis are reported side effects, but the underlying pathophysiological mechanism has not been unravelled. METHODS We studied mitochondrial ultrastructure, mitochondrial respiratory chain enzyme activity, and mitochondrial DNA (mtDNA) in muscle, liver, and kidney samples obtained from a patient who developed optic neuropathy, encephalopathy, skeletal myopathy, lactic acidosis, and renal failure after prolonged use of linezolid. In addition, we evaluated mtDNA, respiratory chain enzyme activity, and protein amount in muscle and liver samples obtained from experimental animals that received linezolid or placebo. RESULTS In the patient, mitochondrial respiratory chain enzyme activity was decreased in affected tissues, without ultrastructural mitochondrial abnormalities and without mutations or depletion of mtDNA. In the experimental animals, linezolid induced a dose- and time-dependent decrease of the activity of respiratory chain complexes containing mtDNA-encoded subunits and a decreased amount of protein of these complexes, whereas the amount of mtDNA was normal. CONCLUSION These results provide direct evidence that linezolid inhibits mitochondrial protein synthesis with potentially severe clinical consequences. Prolonged courses of linezolid should be avoided if alternative treatment options are available.

Quantification of Expression of Staphylococcus epidermidis Housekeeping Genes with Taqman Quantitative PCR during In Vitro Growth and under Different Conditions

The aims of the present study were (i) to develop and test a sensitive and reproducible method for the study of gene expression in staphylococci and (ii) to study the expression of five housekeeping genes which are involved in nucleic acid metabolism (gmk, guanylate kinase; the dihydrofolate reductase [DHFR] gene), glucose metabolism (tpi, triosephosphate isomerase), and protein metabolism (the 16S rRNA gene; hsp-60, heat-shock protein 60) during in vitro exponential and stationary growth. A modified method for instant mRNA isolation was combined with gene quantification via Taqman real-time quantitative PCR. The detection limit of our method was 10 copies of RNA. The average intersample variability was 16%. A 10-fold increase in the expression of the hsp-60 gene was induced by exposure to a 10 degrees C heat shock (37 to 47 degrees C) for 10 min. During in vitro growth, the expression of all five housekeeping genes showed rapid up-regulation after inoculation of the bacteria in brain heart infusion medum and started to decline during the mid-exponential-growth phase. Maximal gene expression was 110- to 300-fold higher than gene expression during stationary phase. This indicates that housekeeping metabolism is a very dynamic process that is extremely capable of adapting to different growth conditions. Expression of the 16S rRNA gene decreases significantly earlier than that of other housekeeping genes. This confirms earlier findings for Escherichia coli that a decline in bacterial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression).

The Effect of the Host's Iron Status on Tuberculosis

Several lines of evidence have suggested that iron is critical for Mycobacterium tuberculosis growth in macrophages. Macrophage iron loading in patients with African iron overload increases the risk of tuberculosis (TB) and may worsen TB outcome. Likewise, macrophage iron loading may contribute to an increased predisposition toward TB in HIV infection. Human genetic disorders or variations may increase the risk of TB or worsen its outcome through macrophage iron loading, including the haptoglobin 2-2 phenotype, NRAMP1 polymorphisms (at least in Africans and Asians), and possibly ferroportin 1 mutations, but not HFE hemochromatosis. Thus, the hosts iron status may be an important yet underevaluated factor in TB prevention and therapy and in TB vaccine design.

Difference in time to positivity of hub-blood versus nonhub-blood cultures is not useful for the diagnosis of catheter-related bloodstream infection in critically ill patients.

ObjectiveThe differential time to positivity (DTTP), defined as the difference in time necessary for the blood cultures taken by a peripheral puncture and through the catheter to become positive has been suggested to be useful in differentiating between catheter-related bloodstream infection (CR-BSI) and other sources of bacteremia. A DTTP of >120 mins was found predominantly in CR-BSI. The objective of our study was to investigate whether DTTP is useful for the diagnosis of CR-BSI in a medical-surgical intensive care unit. DesignProspective clinical study. SettingA 60-bed medical-surgical intensive care unit of a university hospital. PatientsOne hundred consecutive adult patients from whom catheter(s) were to be removed for suspected CR-BSI were studied. InterventionA blood culture (using aerobic and anaerobic culture bottles) was first taken from a new puncture site. Next, a blood culture was taken through every intravascular catheter in place. Measurements and Results DTTP was calculated using the automated BacT/Alert blood culture system. Three patients had CR-BSI and nine patients had noncatheter-related bacteremia. Five patients had catheter-related sepsis without proven bacteremia. There was no significant difference in median DTTP between patients with CR-BSI and noncatheter-related bacteremia (2.1 hrs and 3.3 hrs, respectively;p = .6). Moreover, catheter-related sepsis in patients without bacteremia could not be detected using DTTP. ConclusionDTTP seems not to be useful for the diagnosis of CR-BSI in a medical-surgical intensive care unit.

Recent changes in vancomycin use in renal failure

Vancomycin is a key tool in the treatment of serious Gram-positive infections. A progressive increase in vancomycin resistance with consequent treatment failure has been observed in staphylococci. Therefore, new dosing guidelines advocating much higher vancomycin doses have been issued. Target trough levels of 15-20 microg/ml are proposed. Whether and how these targets can be achieved in patients with chronic kidney disease or those on dialysis are still under evaluation. The higher vancomycin doses to achieve these treatment targets carry a substantial risk for nephrotoxicity. This risk is incremental with higher trough levels and longer duration of vancomycin use. Critically ill patients, patients receiving concomitant nephrotoxic agents, and patients with already compromised renal function are particularly at risk for vancomycin-induced nephrotoxicity.

The pharmacokinetics and pharmacodynamics of vancomycin in clinical practice: evidence and uncertainties

Vancomycin has been used extensively since the late 1950s. Despite the introduction of several new valuable anti-Gram-positive antibiotics during recent years and the waning susceptibility of staphylococci to vancomycin, it remains the gold standard for the treatment of bacteraemia caused by methicillin-resistant staphylococci. Vancomycin has clear dose-response and dose-toxicity correlations. It is widely accepted that these correlations are best predicted by the AUC/MIC model, with target levels of >400 being the clinical cut-off. The experimental base of this model is less robust than frequently believed, and several important issues in vancomycin resistance, such as biofilm resistance and the inoculum effect, are not included. Based on this model, current dosing guidelines propose intermittent dosing of vancomycin with target trough levels of 15-20 mg/L. Dose adaptations according to renal function have been proposed but are not yet validated. Clinical data also support the use of continuous infusion with target plateau levels of 20-25 mg/L, with similar efficacy at the cost of lower nephrotoxicity. Despite decades of intense clinical use and numerous studies and publications, the optimal dosing strategy for vancomycin reconciling the high needs of the dose-response relationship with the serious drawbacks of the dose-toxicity relationship remains to be established.

Diffuse Alveolar Hemorrhage Induced by Everolimus

Pulmonary toxicity is a known complication of the proliferation signal inhibitor (PSI) sirolimus and consists of diverse entities such as interstitial pneumonitis, lymphocytic alveolitis, bronchiolitis obliterans with organizing pneumonia, and diffuse alveolar hemorrhage. Several cases of interstitial pneumonitis have also been reported with the more recently developed PSI everolimus. In this report, a case of diffuse alveolar hemorrhage attributed to everolimus is described. The patient presented with respiratory symptoms of insidious onset, ultimately resulting in severe respiratory failure characterized by high lactate dehydrogenase levels, patchy ground-glass infiltrates, and bloody BAL fluid with predominance of iron-loaded macrophages and monocytes. Withdrawal of the offending drug and temporary association of high-dose steroids resulted in a rapid recovery. Given that prompt drug discontinuation is potentially life saving, PSI-induced pulmonary toxicity should be considered in the differential diagnosis of patients treated with PSIs and presenting with respiratory symptoms or pulmonary lesions.

Vancomycin Dosing in Patients on Intermittent Hemodialysis

Vancomycin has been a cornerstone antibiotic for the treatment of severe gram‐positive infections in dialysis patients for decades. Whereas subtherapeutic vancomycin levels convey a risk of treatment failure and the further emergence of resistance in staphylococci, supratherapeutic vancomycin levels are associated with a dose‐related incremental risk for nephrotoxicity and ototoxicity. Consequently, a narrow therapeutic range with a trough‐level target between 15 and 20 μg/ml is recommended. Vancomycin dosing in hemodialysis patients is mainly influenced by the timing of administration (during or after dialysis), the type of filter used, and the duration of dialysis. Actual body weight, the interdialytic interval, and residual renal function are also considerations. As in patients with normal kidney function, a weight‐based loading dose of 20–25 mg/kg should be used in dialysis patients. While most fixed‐dose maintenance regimens fail to reach target levels in the majority of hemodialysis patients, straightforward evidence on optimal maintenance dosing is lacking.

Vitamin K2 supplementation in haemodialysis patients: a randomized dose-finding study

BACKGROUND Haemodialysis patients suffer from accelerated vascular calcification. The vitamin K-dependent matrix Gla protein (MGP) is one of the most powerful inhibitors of vascular calcification. Haemodialysis patients have high levels of the inactive form of MGP (desphosphorylated-uncarboxylated-MGP, dp-uc-MGP) and may benefit from pharmacological doses of vitamin K2 (menaquinone) to improve the calcification inhibitory activity of MGP. METHODS To determine the optimal dose of menaquinone-7 (MK-7) for MGP activation, 200 chronic haemodialysis patients were recruited to randomly receive 360, 720 or 1080 µg of MK-7 thrice weekly for 8 weeks. Dp-uc-MGP was measured at baseline and after 8 weeks. Dietary intake of vitamin K1 (phylloquinone) and menaquinone was estimated based on a detailed questionnaire. RESULTS At baseline, dp-uc-MGP was not associated with phylloquinone intake (P = 0.92), but correlated inversely with menaquinone intake (P = 0.023). MK-7 supplementation dose dependently reduced dp-uc-MGP. The levels decreased by 17, 33 and 46% in the respective groups. Drop-outs were mainly due to gastrointestinal side-effects related to the unpleasant smell of the tablets. CONCLUSIONS Chronic haemodialysis patients have high levels of inactive MGP, possibly related to a low dietary vitamin K intake. Pharmacological doses of MK-7 dose-dependently reduce dp-uc-MGP. Menaquinone supplementation may be a novel approach to prevent vascular calcifications in chronic haemodialysis patients.

The role of σB in persistence of Staphylococcus epidermidis foreign body infection

Staphylococcal biofilm formation depends on the transcription factor sigma(B). We further investigated the role of sigma(B) in biofilm formation and persistence in vitro and in vivo in a subcutaneous rat model. As expected, expression of all sigma(B) operon genes was transiently higher in the first 6 h of biofilm formation compared to planktonic bacteria, concurrent with a temporary upregulation of icaA and aap expression. However, we also observed a second upregulation of sigB expression in biofilm more than 2 days old without upregulation of icaA or aap. Biofilm formation by Staphylococcus epidermidis strains 8400 and 1457 was compared to that of isogenic mutants with inactivation of rsbU, of rsbUVW and of the entire sigma(B) operon. Both wild-type strains and the constitutively sigB-expressing rsbUVW mutant showed a strong biofilm-positive phenotype. The rsbUVW mutant biofilm was, however, thinner and more evenly spread than the wild-type biofilm. Inactivation of SigB in the rsbUVWsigB mutant or mutation of the positive regulator RsbU reduced both the number of sessile bacteria and polysaccharide intercellular adhesin (PIA) synthesis. These differences between the wild-types and their respective mutants appeared after 6 h in in vitro biofilms but only after 4 days in in vivo biofilms. Our results provide additional evidence for a role for sigma(B) in biofilm formation. They also suggest a role for sigma(B) in biofilm maturation and stability that is independent of PIA or accumulation-associated protein (Aap) and point to significant differences in the temporal development between in vitro and in vivo biofilms.